Affinity reagents having enhanced binding and detection characteristics

ABSTRACT

An affinity reagent, having: (a) a retaining component such as a structured nucleic acid particle; and (b) one or both of (i) one or more label components attached to the retaining component, and (ii) one or more binding components attached to the retaining component.

CROSS-REFERENCE

This application is a continuation of U.S. application Ser. No.17/523,869, filed Nov. 10, 2021, which claims the benefit of priority toU.S. Provisional Application No. 63/112,607, filed on Nov. 11, 2020,U.S. Provisional Application No. 63/132,170, filed on Dec. 30, 2020, andU.S. Provisional Application No. 63/227,080, filed on Jul. 29, 2021,each of which is incorporated herein in its entirety by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in XML format and is hereby incorporated byreference in its entirety. Said XML copy, created on Mar. 1, 2023, isnamed 50109_4003.xml and is 22,530 bytes in size.

BACKGROUND

Affinity reagents include a broad class of chemical reagents that formdetectable interactions with other molecules. Affinity reagents mayinclude binding reagents that form temporary or reversible binding pairswith other molecules. Affinity reagents may be utilized to characterizethe structure and properties of biomolecules, such as polypeptides,nucleic acids, and polysaccharides. Affinity reagents may includedetectable labels for the purpose of visualizing the affinity reagent.Frequently these detectable labels are fluorescent labels. There is aneed for labeled affinity reagents that produce strong and reliablesignals including, for example, signals strong enough for singlemolecule detection, and reliable enough for accurate quantification.There is also a need for affinity reagents that bind avidly to targetmolecules, for example, with sufficient avidity to support detection oftarget molecules at single molecule resolution.

SUMMARY

The present disclosure provides an affinity reagent, having: (a) aretaining component; and (b) one or both of (i) one or more labelcomponents, and (ii) one or more binding components. Optionally, one ormore of the label components is attached to the retaining component. Asan alternative or additional option, one or more of the bindingcomponents is attached to the retaining component. The retainingcomponent can include a structured nucleic acid such as a nucleic acidorigami.

An affinity reagent can include: (a) a retaining component; (b) one ormore label components, and (ii) a plurality of binding components.Optionally, the affinity reagent has an equilibrium dissociationconstant that is less than the equilibrium dissociation constant of anyone of the plurality binding components for the binding partner, orwherein the detectable probe has a dissociation rate constant that isless than the dissociation rate constant of any of the plurality ofbinding components for the binding partner.

An affinity reagent can include: (a) a retaining component; (b) aplurality of label components, and (ii) one or more binding components.

An affinity reagent of the present disclosure can be configured as adetectable probe. A detectable probe can include: (a) a retainingcomponent; (b) one or more label components; and (c) two or more bindingcomponents attached to the retaining component, wherein the detectableprobe has an equilibrium dissociation constant for a binding partnerthat is less than the equilibrium dissociation constant of any of thetwo or more binding components for the binding partner, or wherein thedetectable probe has a dissociation rate constant for a binding partnerthat is less than the dissociation rate constant of any of the two ormore binding components for the binding partner.

The present disclosure further provides a method of detecting ananalyte, including the steps of: (a) contacting an analyte with adetectable probe, wherein the detectable probe comprises (i) a retainingcomponent; (ii) one or more label components, and (iii) one or morebinding components; and (b) acquiring a signal from the one or morelabel components, thereby detecting the analyte.

Also provided is a method of detecting an analyte, including steps of(a) contacting an analyte with a first detectable probe, the firstdetectable probe including: (i) a first retaining component, (ii) one ormore label components, and (iii) a first set of two or more bindingcomponents attached to the retaining component, wherein at least one ofthe binding components in the first set binds to a first epitope in theanalyte; (b) acquiring a signal from the one or more label components ofthe first detectable probe; (c) contacting the analyte with a seconddetectable probe, the second detectable probe including: (i) a secondretaining component (ii) one or more label components, and (iii) asecond set of two or more binding components attached to the retainingcomponent, wherein at least one of the binding components in the secondset binds to a second epitope in the analyte, the second epitope havinga different chemical composition compared to the first epitope; and (d)acquiring a signal from the one or more label components of the seconddetectable probe, thereby detecting the analyte. Optionally, the secondretaining component has a structure that is substantially the same asthe first retaining component.

The present disclosure also provides a composition, including: (a) aprobe having a first structured nucleic acid particle attached to abinding component; and (b) an analyte having a second structured nucleicacid particle attached to an epitope for the binding component, whereinthe probe is attached to the analyte via binding of the bindingcomponent to the epitope. In particular configurations one or both ofthe structured nucleic acid particles include nucleic acid origami.

Also provided is a composition, including: (a) a plurality of differentprobes, each of the different probes having a first structured nucleicacid particle attached to a binding component, each of the differentprobes having a different binding component; and (b) a plurality ofdifferent analytes, each of the different analytes having a secondstructured nucleic acid particle attached to an epitope for a differentbinding component, wherein the different probes are attached to thedifferent analytes via binding of a different binding component of theplurality of different probes to an epitope of the plurality ofdifferent analytes. Optionally, the first structured nucleic acidparticle is substantially the same for the different probes. As afurther option, the second structured nucleic acid particle can besubstantially the same for the different analytes. In particularconfigurations one or both of the structured nucleic acid particlesinclude nucleic acid origami. The nucleic acid origami of the differentprobes can optionally include the same scaffold nucleic acid structure,whether or not staple structures are the same or different for thedifferent probes. Alternatively or additionally, the nucleic acidorigami of the different analytes can optionally include the samescaffold nucleic acid structure, whether or not staple structures arethe same or different for the different analytes.

Described herein is a detectable probe comprising a retaining componentincluding one or more label components (e.g. detectable labels), and twoor more binding components coupled to the retaining component, where thedetectable probe has a dissociation constant for a binding partner thatis less than a dissociation constant of any of the two or more bindingcomponents for the binding partner.

In some configurations, one or more label components of a detectableprobe are coupled to the retaining component of the detectable probe. Insome embodiments, the retaining component includes a scaffold having aclosed single-stranded nucleic acid, and a plurality of oligonucleotideshybridized to the scaffold.

In some configurations, a retaining component in a detectable probe oraffinity reagent includes a scaffold nucleic acid. A scaffold caninclude a strand from a phage genome or a plasmid, for example, from anM13 phage genome. Optionally, a retaining component can further includea plurality of oligonucleotides. The oligonucleotides can be annealed tothe scaffold, for example, to form staples in an origami structure. Insome configurations, an oligonucleotide of the plurality ofoligonucleotides can include at least one non-natural nucleotide.Optionally, a non-natural nucleotide in an oligonucleotide can have afunctional group, such as a functional group used in a bioorthogonal orclick reaction. In some configurations, one, two or more bindingcomponents are attached to one or more oligonucleotides of the pluralityof oligonucleotides. In some configurations, one, two or more labelcomponents are attached to one or more oligonucleotides of the pluralityof oligonucleotides. In some configurations, a scaffold can include atleast one non-natural nucleotide. Optionally, a non-natural nucleotidein a scaffold can have a functional group, such as a functional groupused in a bioorthogonal or click reaction. In some configurations, one,two or more binding components are attached to a scaffold. In someconfigurations, one, two or more label components are attached to ascaffold.

In some configurations, a detectable probe or affinity regent having twoor more binding components has a dissociation constant for a bindingpartner that is less than the dissociation constant for any of the twoor more binding components for the binding partner. For example, thedetectable probe or affinity regent can have a dissociation constant fora binding partner that is less than or equal to 50%, 25%, 10% or less ofthe dissociation constant for any of the two or more binding componentsfor the binding partner. In some configurations, the off-rate of adetectable probe or affinity reagent when bound to a binding partner islower than an individual off-rate for any of the two or more bindingcomponents when bound to a binding partner. In some configurations, theon-rate of a detectable probe or affinity reagent for a binding partneris higher than an individual on-rate for any of the two or more bindingcomponents for the binding partner.

In some configurations, a detectable probe or affinity reagent hasnon-zero binding affinity for a first type of epitope and non-zerobinding affinity for a second type of epitope. For example, thedetectable probe or affinity reagent can have a first non-zero bindingprobability to a first type of epitope and a second non-zero bindingprobability to a second type of epitope. In some configurations, a firstbinding component of two or more binding components of the detectableprobe or affinity reagent has the first non-zero binding probability tothe first type of epitope and also has the second non-zero bindingprobability to the second type of target moiety. In otherconfigurations, a first binding component of the two or more bindingcomponents includes the first non-zero binding probability to the firsttype of target moiety, and a second binding component of the two or morebinding components includes the second non-zero binding probability tothe second type of target moiety.

In some configurations, at least one of the binding components in adetectable probe or affinity reagent includes an antibody or functionalfragment thereof, wherein a binding partner of the detectable probe oraffinity reagent has an epitope for the antibody or functional fragmentthereof. In some configurations, at least one of the binding componentsin a detectable probe or affinity reagent includes an aptamer, wherein abinding partner of the detectable probe or affinity reagent has anepitope for the aptamer.

In some configurations, a detectable probe or affinity reagent is boundto a binding partner via at least one binding component. Optionally, thebinding partner can bound to the detectable probe or affinity reagentvia two or more binding components. The binding partner can be apolypeptide. At least one of the binding components can be configured torecognize dimer, trimer or tetramer amino acid sequences inpolypeptides. Optionally, the polypeptide can include apost-translational modification, for example, present within an epitoperecognized by a binding component or outside of the epitope. In someembodiments, the binding partner includes a non-polypeptide materialsuch as a polysaccharide, polymer, metal, ceramic, or a combinationthereof. In some embodiments, the non-polypeptide material includes ananoparticle of a polysaccharide, polymer, metal, or ceramic. Adetectable probe or affinity reagent can be non-covalently bound to abinding partner or covalently bound to a binding partner. A bindingpartner that is bound to a detectable probe or affinity reagent, can bein solution phase or attached to a solid support. Optionally, thebinding partner can be attached to a structured nucleic acid particleother than a structured nucleic acid particle that is a component of thedetectable probe or affinity reagent to which it binds. A structurednucleic acid particle can optionally mediate attachment of a bindingpartner to a solid support, for example, at a site of an array.

A retaining component of a detectable probe or affinity reagent havingtwo or more binding components can be configured to constrain a firstbinding component of the two or more binding components from contactinga second binding component of the two or more binding components. Aretaining component of a detectable probe or affinity reagent having twoor more label components can be configured to constrain a first labelcomponent of the two or more label components from contacting a secondlabel component of the two or more label components. A retainingcomponent of a detectable probe or affinity reagent having a labelcomponent and a binding component can be configured to constrain thelabel component from contacting the binding component. Optionally, aretaining component can constrain a first component of two or morecomponents from coming within a specified distance of a second componentof the two or more components, for example, a distance of no more than 1nm, 5 nm, 10 nm, 20 nm or more. In some configurations, the constraintis due to an angular offset. For example, the angular offset can be atleast about 90° or 180°. In some configurations, the constraint is dueto a blocking moiety, for example, a blocking moiety attached to theretaining component. Exemplary blocking moieties include but are notlimited to polyethylene glycol (PEG), polyethylene oxide (PEO), linearor. branched alkane chains, or dextran.

A retaining component of a detectable probe or affinity reagent caninclude a three-dimensional structure having a first side that is offsetfrom a second side. For example, the first side can have an offset fromthe second side that is an angular offset of at least about 90° or 180°.Optionally, one, some or all binding components of a detectable probe oraffinity reagent are constrained to residing on the first side andconstrained from residing on the second side. In some configurations,the first side and the second side can each include one or more bindingcomponents. As a further option, one, some or all label components of adetectable probe or affinity reagent can be constrained to residing onthe first side of a retaining component and constrained from residing ona second side of the retaining component. In some configurations, thefirst side and the second side can each include one or more labelcomponents. Accordingly, a detectable probe or affinity reagent can beconfigured to retain binding components on a first side of a retainingcomponent while retaining label components on another side of theretaining component. A detectable probe or affinity reagent can beconfigured to constrain one, some, or all binding components fromresiding on the side of a retaining component where one, some, or alllabel components reside. Moreover, a detectable probe or affinityreagent can be configured to constrain one, some, or all labelcomponents from residing on the side of a retaining component where one,some, or all binding components reside. A retaining component of adetectable probe or affinity reagent can include a structured nucleicacid particle (SNAP) such as a nucleic acid nanoball or nucleic acidorigami.

One or more label components of a detectable probe or affinity reagentcan include any of a variety of labels including, for example, opticallabels (e.g. fluorophore, luminophore), radiolabels, or nucleic acidbased labels (e.g. sequence tags). In some configurations, two or morelabel components of a detectable probe or affinity reagent can produceoverlapping or indistinguishable signals. For example, two or more labelcomponents can be fluorophores that are configured to emit at the samewavelength. In some configurations, two or more label components of adetectable probe or affinity reagent produce signals that are resolvedfrom each other. For example, two or more label components can befluorophores that are configured to emit at different wavelengths fromeach other.

In some configurations, two or more label components of a detectableprobe or affinity reagent include a donor and acceptor in a Forsterresonant energy transfer mechanism. Alternatively, two or more labelcomponents of a detectable probe or affinity reagent can be separatedfrom each other by a distance that precludes quenching or Forsterresonant energy transfer. Two or more label components of a detectableprobe or affinity reagent can have a relative orientation that precludesquenching or Forster resonant energy transfer. For configurations thatinclude a SNAP, a first fluorescent label can be attached at a firstnucleotide position in the SNAP and a second fluorescent label isattached at a second nucleotide position in the SNAP, wherein the firstnucleotide position is separated from the second nucleotide position byat least 3, 4, 5, 6, 7, 8 or 9 nucleotide positions in the primarysequence of the structured nucleic acid particle. Alternatively oradditionally, the first nucleotide position can be separated from thesecond nucleotide position by at most 9, 8, 7, 6, 5, 4, 3, 2, or 1nucleotide positions in the primary sequence of the structured nucleicacid particle.

A binding component or label component can be attached to a detectableprobe or affinity reagent by a linker. The linker can be a rigid linkeror a flexible linker. Exemplary flexible linkers include, but are notlimited to, PEG, PEO, an alkane chain, a single stranded nucleic acid,or a combination thereof. A double-stranded nucleic acid or branchedalkane chain can be used as a rigid linker.

In some configurations, the major diameter of a detectable probe oraffinity reagent is larger than the major diameter of a binding partnerto which it binds. Optionally, the volume of a detectable probe oraffinity reagent is larger than the volume of a binding partner to whichit binds.

In particular configurations, a detectable probe or affinity reagent caninclude an optically detectable retaining component. For example, one,two or more binding components can be attached to theoptically-detectable retaining component. In such configurations, thedetectable probe or affinity reagent need not include label componentsand detection can be carried out based on observation of signalsproduced by the optically detectable retaining component. Particularlyuseful optically detectable retaining components include, but are notlimited to, fluorescent nanoparticles, FluoSpheres™, or quantum dots.

In another aspect, described herein is a detectable probe or affinityreagent including an optically detectable retaining component, and twoor more binding components coupled to the optically-detectable retainingcomponent, where the detectable probe or affinity reagent has adissociation constant for a binding partner that is less than thedissociation constant of any of the two or more binding components forthe binding partner.

In another aspect, described herein is a detectable probe or affinityreagent including an optically detectable retaining component, and twoor more binding components coupled to the optically-detectable retainingcomponent, where the detectable probe or affinity reagent has a bindingon-rate for a binding partner that is higher than the binding on-rate ofany of the two or more binding components for the binding partner.

In another aspect, described herein is a detectable probe or affinityreagent including an optically detectable retaining component, and twoor more binding components coupled to the optically-detectable retainingcomponent, where the detectable probe or affinity reagent has a bindingoff-rate for a binding partner that is lower than the binding off-rateof any of the two or more binding components for the binding partner.

In another aspect, described herein is a method including contacting ananalyte with a detectable probe, wherein the analyte is a bindingpartner of the detectable probe, and acquiring a signal from thedetectable probe, thereby detecting the analyte. The method can furtherinclude identifying the analyte from the acquired signal, or determiningthe chemical composition of the analyte from the acquired signal. Insome embodiments, the analyte includes a polypeptide and the chemicalcomposition that is determined includes the presence or absence of anamino acid sequence of at least a portion of the polypeptide, or thepresence or absence of a post translationally modified amino acid in thepolypeptide. In some embodiments, the method further includesquantifying the analyte from the acquired signal. In some embodiments,the method further includes determining the location of the analyte on asolid support from the acquired signal, for example, identifying a sitein an array where the analyte resides. In some embodiments, the signalis acquired from an optically detectable retaining component of thedetectable probe. In some embodiments, the signal is acquired from oneor more label components of the detectable probe.

In another aspect, described herein is a method including (a) contactinga plurality of different analytes with a first plurality of detectableprobes, wherein detectable probes from the first plurality of detectableprobes bind to a first subset of different analytes from the pluralityof different analytes, (b) acquiring signals from the first plurality ofdetectable probes, (c) contacting the plurality of different analyteswith a second plurality of detectable probes, wherein detectable probesfrom the second plurality of detectable probes bind to a second subsetof different analytes from the plurality of different analytes, wherethe first subset of different analytes is different from the secondsubset of analytes, (d) acquiring signals from the second plurality ofdetectable probes, and (e) identifying analytes based on the signalsacquired in step (b) and step (d). Optionally, the first plurality ofdetectable probes include substantially the same two or more bindingcomponents as the second plurality of detectable probes. Alternatively,the first plurality of detectable probes can include two or more bindingcomponents that are different from the two or more binding components ofthe second plurality of detectable probes. As a further option, one,some or all the detectable probes in the first plurality can include aretaining component. Optionally, the retaining components can have acommon structure for some or all the detectable probes in the firstplurality. For example, the retaining components for some or all thedetectable probes can include an origami structure, such as a scaffoldfolding structure, that is the same. Similarly, the retaining componentfor one, some or all the detectable probes in the first plurality canhave a structure in common with the retaining component for one, some orall the detectable probes in the second plurality. For example, theretaining components for some or all the detectable probes in the firstand second pluralities can include an origami structure, such as ascaffold folding structure, that is the same.

In some configurations, the above method can further include a step ofremoving the first plurality of detectable probes from the plurality ofdifferent analytes prior to step (c). In some configurations of theabove method, the first plurality of detectable probes produce the samesignal as a signal produced one or more label components as the secondplurality of detectable probes. For example, the first plurality ofdetectable probes can include the same one or more label components asthe second plurality of detectable probes. In some configurations of theabove method, the first plurality of detectable probes include one ormore label components that are different from the one or more labelcomponents of the second plurality of detectable probes. In someconfigurations of the above method, the first plurality of detectableprobes include the same optically detectable retaining component as thesecond plurality of detectable probes. In some configurations of theabove method, the first plurality of detectable probes include adifferent optically detectable retaining component from the opticallydetectable retaining component of the second plurality of detectableprobes.

One or more different analytes that are bound to an affinity agent ordetected by a detectable probe in a method set forth herein can beattached to a solid support. For example, individual analytes of aplurality of different analytes can be attached at respective sites on asolid support, whereby the solid support includes an array of analytes.

Further provided by the present disclosure is a method of locating abinding partner, including (a) providing a material (e.g. a solidsupport) including a binding partner at a discrete location in thematerial; (b) contacting the material with a detectable probe, whereinthe detectable probe includes (i) a retaining component, (ii) one ormore label components that are configured to produce a detectablesignal, and (iii) two or more binding components coupled to theretaining component, wherein the retaining component and two or morebinding components form a detectable probe; (c) detecting the detectablesignals from the one or more label components; and (d) identifying thediscrete location of the detectable signal, thereby locating the bindingpartner in the material. Optionally, the detectable probe binds thebinding partner with a dissociation constant that is less than or equalto half of a dissociation constant for binding of the binding partner toany of the binding components, individually.

Also provided is a method of forming a detectable probe or affinityreagent, including (a) providing a retaining component including aplurality of coupling groups, and (b) attaching coupling groups of theplurality of coupling groups to a plurality of binding components.Optionally, the method further includes (c) attaching coupling groups ofthe plurality of coupling groups to a plurality of label components.

Attachment between components of a detectable probe or affinity reagentcan be covalent or non-covalent. Nucleic acids provide a particularlyuseful coupling group. For example, the coupling groups in the abovemethod can include single-stranded nucleic acid moieties that anneal tocomplementary nucleic acids attached to the binding components and/orlabel components. The retaining component can include nucleic acidorigami that is engineered to position the single-stranded nucleic acidmoieties at known positions. For example, the single-stranded nucleicacid moieties can be included in staple strands or oligonucleotides thatare annealed to a scaffold strand. Other useful non-covalent attachmentsinclude, for example, those mediated by receptor-ligand pairs such asstreptavidin-biotin, SpyCatcher-SpyTag, SdyCatcher-SdyTag, andSnoopCatcher-SnoopTag.

Any of a variety of functional groups can be used to covalently attachcomponents of a detectable probe or affinity reagent. Bioorthogonalreactions and Click reactions are particularly useful.

A method for making a detectable probe or affinity reagent can include astep of forming a passivating layer on a retaining component.Passivation of a retaining component can be carried out, for example,prior to attaching functional groups (e.g. coupling groups), labelcomponents or binding components to the retaining component. Optionally,the passivating layer can include a metal, metal oxide, organicfunctional group, or polymer. For example, the metal can include gold,silver, copper, titanium, or iron. Optionally, the metal oxide includestitanium oxide, alumina, silicon dioxide, or magnesium oxide.Optionally, the organic functional group includes a phosphate, aphosphonate, a carboxylate, an epoxide, or a silane. In someembodiments, the polymer includes a hydrocarbon polymer or a biopolymer.In some embodiments, the hydrocarbon polymer includes polyethyleneglycol (PEG), polyethylene oxide (PEO), or an alkane chain. In someembodiments, the biopolymer includes a polysaccharide, a polynucleotide,or a polypeptide.

A detectable probe of the present disclosure can include a doublestranded region and an aptamer; wherein the double stranded regionincludes two or more label components. For example, the label componentscan be fluorescently labeled nucleotides in one or both of the strands.Optionally, the fluorescently labeled nucleotide can be separated fromeach other, for example, by at least 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,35, 40, 45, 50 or more nucleotides. Optionally, the double strandedregion can include at least 3, 4, 5, 6, 7, 8, 9, 10 or more fluorophoresor other label components. Two or more label components of a detectableprobe can be the same as each other or different from each other.

Optionally an aptamer, or other binding component, that is included in adetectable probe or affinity reagent can recognize or bind to some orall sequences of the form αXβ, wherein X is a desired epitope and α andβ are any amino acid residues. Optionally, the aptamer can recognize orbind to at least 10%, 20%, 30%, 50%, 80% or 90% of sequences of the formαXβ.

In some configurations, an aptamer or other binding component, that isincluded in a detectable probe or affinity reagent can recognize or bindto a desired three amino acid epitope, without specifically binding anyother three amino acid sequences, and binds the desired three amino acidepitope with substantially similar affinity regardless of flankingsequence surrounding the desired epitope.

In another aspect, described herein is a switchable aptamer which bindsto between 5% and 10% of all proteins in the human proteome; and whereinthe switchable aptamer includes two or more fluorescent moieties.

The present disclosure further provides a method of manufacturing afluorescently labeled aptamer, the method including synthesizing anaptamer with a primer sequence at the 3′ end, hybridizing a template DNAstrand to the primer sequence, wherein the template DNA strand includesa segment complementary to the primer and a template region, and using apolymerase to extend the 3′ end of the aptamer molecule along thetemplate. Optionally, the polymerase reaction is performed with anucleotide mix including labeled nucleotides. For example, one or morespecies of nucleotide can include a label, whereas the other species ofnucleotide lack labels. For example the polymerase reaction can becarried out with four nucleotides (adenine, cytosine, guanine, andthymine or uracil) of which three nucleotides are non-labeled and thefourth nucleotide is fluorescently labeled, and wherein the template isdesigned such that the base complementary to the fluorescently labelednucleotide occurs in a predetermined pattern. Optionally, the basecomplementary to the fluorescently labeled nucleotide can occur in every2^(nd) 3^(rd) 4^(th), 5^(th), 6^(th), 7^(th), 8^(th), 9^(th), 10^(th),15^(th), 20^(th), 25^(th), 30^(th), 35^(th), 40^(th), 45^(th), or50^(th) position along the template.

In another aspect, described herein is a method of manufacturing theabove-described fluorescently labeled aptamer, the method includingligating an aptamer to a fluorescently labeled oligonucleotide. Inanother configuration, a method of manufacturing a fluorescently labeledaptamer, can include synthesizing an aptamer with an extension sequenceat the 3′ end, hybridizing a splint nucleic acid strand to the extensionsequence, and hybridizing a labeled oligonucleotide to the splintnucleic acid strand such that an end of the labeled oligonucleotide isadjacent to an end of the extension sequence, and using a ligase toligate the labeled oligonucleotide to the aptamer via the extensionsequence.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows an object with an angular offset between two faces of theobject.

FIG. 1B shows an object with an angular offset between two faces of theobject.

FIG. 2A shows an approximate two-dimensional projection of a squareretaining component.

FIG. 2B shows an approximate two-dimensional projection of a circularretaining component.

FIG. 3A shows relative separation between two binding components.

FIG. 3B shows relative separation between two binding components.

FIG. 3C shows relative separation between two binding components.

FIG. 4A shows configurations for separating two binding components.

FIG. 4B shows configurations for separating two binding components.

FIG. 4C shows configurations for separating two binding components.

FIG. 4D shows configurations for separating two binding components.

FIG. 5A shows configurations for spacing label components on a retainingcomponent.

FIG. 5B shows configurations for spacing label components on a retainingcomponent.

FIG. 6A shows a configuration of binding components and label componentson a retaining component.

FIG. 6B shows a configuration of binding components and label componentson a retaining component.

FIG. 6C shows a configuration of binding components and label componentson a retaining component.

FIG. 6D shows a configuration of binding components and label componentson a retaining component.

FIG. 6E shows a configuration of binding components and label componentson a retaining component.

FIG. 6F shows a configuration of binding components and label componentson a retaining component.

FIG. 7A shows a configuration of a detectable probe including linkersfor binding components.

FIG. 7B shows a configuration of a detectable probe including linkersfor binding components.

FIG. 8 shows a configuration of a detectable probe with an additionalavidity component.

FIG. 9A shows a configuration of a system for creating a weak bindinginteraction for a detectable probe.

FIG. 9B shows a configuration of a system for creating a weak bindinginteraction for a detectable probe.

FIG. 10A shows a method for creating a weak binding interaction for adetectable probe.

FIG. 10B shows a method for creating a weak binding interaction for adetectable probe.

FIG. 10C shows a method for creating a weak binding interaction for adetectable probe.

FIG. 11A shows a method for creating a weak binding interaction with twodetectable probes.

FIG. 11B shows a method for creating a weak binding interaction with twodetectable probes.

FIG. 11C shows a method for creating a weak binding interaction with twodetectable probes.

FIG. 11D shows a method for creating a weak binding interaction with twodetectable probes.

FIG. 11E shows a method for creating a weak binding interaction with twodetectable probes.

FIG. 12A shows fluorescence intensity measurements for a group of sensorpixels for a low-intensity fluorescent label.

FIG. 12B shows fluorescence intensity measurements for a group of sensorpixels for a high-intensity fluorescent label.

FIG. 13 shows a cross-sectional view of a highly observable detectableprobe binding with a binding partner.

FIG. 14A shows a configuration for creating a Fluorescent resonanceenergy transfer (FRET) pair on a retaining component.

FIG. 14B shows a configuration for creating a Fluorescent resonanceenergy transfer (FRET) pair on a detectable probe.

FIG. 15A shows a first step of a method for detecting a bindinginteraction utilizing a nucleic acid barcode and a weak secondaryinteraction.

FIG. 15B shows a second step of a method for detecting a bindinginteraction utilizing a nucleic acid barcode and a weak secondaryinteraction.

FIG. 15C shows a third step of a method for detecting a bindinginteraction utilizing a nucleic acid barcode and a weak secondaryinteraction.

FIG. 15D shows a fourth step of a method for detecting a bindinginteraction utilizing a nucleic acid barcode and a weak secondaryinteraction.

FIG. 16A shows a detectable probe composition including a bindingcompetitor.

FIG. 16B shows a detectable probe composition including a bindingcompetitor.

FIG. 17A shows a method of utilizing a detectable probe or affinityreagent as a capture agent.

FIG. 17B shows a method of utilizing a detectable probe or affinityreagent as a capture agent.

FIG. 17C shows a method of utilizing a detectable probe or affinityreagent as a capture agent for a polypeptide assay.

FIG. 18A shows a first step of a method of detecting the binding of morethan one detectable probe to a binding partner.

FIG. 18B shows a second step of a method of detecting the binding ofmore than one detectable probe to a binding partner.

FIG. 18C shows a third step of a method of detecting the binding of morethan one detectable probe to a binding partner.

FIG. 19A shows a method of utilizing a detectable probe to determine apeptide sequence.

FIG. 19B shows a method of utilizing a detectable probe to determine apeptide sequence.

FIG. 19C shows a method of utilizing a detectable probe to determine apeptide sequence.

FIG. 19D shows a method of utilizing a detectable probe to determine apeptide sequence.

FIG. 20A shows a method of altering a binding interaction utilizing abinding competitor.

FIG. 20B shows a method of altering a binding interaction utilizing abinding competitor.

FIG. 21A shows a method of fabricating a detectable probe.

FIG. 21B shows a method of fabricating a detectable probe.

FIG. 22 shows the use of detectable probes for characterization of aplurality of binding partners.

FIG. 23 shows a simplified schematic of a detectable probe or affinityreagent including a DNA origami retaining component.

FIG. 24A shows binding characterization data for quantum dot-baseddetectable probes.

FIG. 24B shows binding characterization data for quantum dot-baseddetectable probes.

FIG. 25 shows a simplified schematic of a detectable probe or affinityreagent including a DNA origami retaining component.

FIG. 26 shows a scheme for attaching an antibody-based binding componentto an origami retaining component via a click reaction.

FIG. 27 shows an image of an SDS-Page gel containingantibody-oligonucleotide conjugates.

FIG. 28A shows binding data for a detectable probe against a polypeptidetarget.

FIG. 28B shows binding data for a detectable probe against a polypeptidetarget.

FIG. 28C shows binding data for a detectable probe against a polypeptidetarget.

FIG. 28D shows binding data for a detectable probe against a polypeptidetarget.

FIG. 29A shows a fluorescent microscope image of detectable probebinding against a negative control polypeptide array.

FIG. 29B shows a fluorescent microscope image of detectable probebinding against a negative control polypeptide array.

FIG. 29C shows a fluorescent microscope image of detectable probebinding against a polypeptide array.

FIG. 30 shows binding data for a detectable probe against a polypeptidetarget.

FIG. 31A shows a configuration of a non-nucleic acid retainingcomponent.

FIG. 31B shows a configuration of a non-nucleic acid retainingcomponent.

FIG. 31C shows a configuration of a non-nucleic acid retainingcomponent.

FIG. 31D shows a configuration of a non-nucleic acid retainingcomponent.

FIG. 32A shows a first step of a method for forming a non-nucleic acidretaining component.

FIG. 32B shows a second step of a method for forming a non-nucleic acidretaining component.

FIG. 32C shows a first step of a method for forming a non-nucleic acidretaining component.

FIG. 32D shows a second step of a method for forming a non-nucleic acidretaining component.

FIG. 32E shows a third step of a method for forming a non-nucleic acidretaining component.

FIG. 33 shows a method of forming a non-nucleic acid retainingcomponent.

FIG. 34A shows a configuration of a detectable probe including anon-nucleic acid retaining component.

FIG. 34B shows a configuration of a detectable probe including anon-nucleic acid retaining component.

FIG. 34C shows a configuration of a detectable probe including anon-nucleic acid retaining component.

FIG. 35A shows a multi-probe complex formed from a plurality ofdetectable probes.

FIG. 35B shows a multi-probe complex formed from a plurality ofdetectable probes.

FIG. 35C shows a multi-probe complex formed from a plurality ofdetectable probes.

FIG. 36A shows a multi-probe complex with a controlled conformation.

FIG. 36B shows a multi-probe complex with a controlled conformation.

FIG. 37A shows a first step of a method for forming a detectable probefrom a non-nucleic acid retaining component.

FIG. 37B shows a second step of a method for forming a detectable probefrom a non-nucleic acid retaining component.

FIG. 37C shows a third step of a method for forming a detectable probefrom a non-nucleic acid retaining component.

FIG. 38A shows the first step of utilizing a detectable probe oraffinity reagent complex for drug delivery.

FIG. 38B shows the second step of utilizing a detectable probe oraffinity reagent complex for drug delivery.

FIG. 38C shows the third step of utilizing a detectable probe oraffinity reagent complex for drug delivery.

FIG. 38D shows the fourth step of utilizing a detectable probe oraffinity reagent complex for drug delivery.

FIG. 39A shows multi-probe complexes formed with a secondary retainingcomponent.

FIG. 39B shows multi-probe complexes formed with a secondary retainingcomponent.

FIG. 40 shows an affinity chromatography system utilizing a detectableprobe or affinity reagent as a capture agent.

FIG. 41 shows a scheme for preparing FluoSphere™-based detectableprobes.

FIG. 42A shows characterization data for FluoSphere™-based detectableprobes.

FIG. 42B shows characterization data for FluoSphere™-based detectableprobes.

FIG. 42C shows characterization data for FluoSphere™-based detectableprobes.

FIG. 42D shows characterization data for FluoSphere™-based detectableprobes.

FIG. 42E shows characterization data for FluoSphere™-based detectableprobes.

FIG. 42F shows characterization data for FluoSphere™-based detectableprobes.

FIG. 43A shows binding characterization data for FluoSphere™-baseddetectable probes.

FIG. 43B shows binding characterization data for FluoSphere™-baseddetectable probes.

FIG. 43C shows binding characterization data for FluoSphere™-baseddetectable probes.

FIG. 43D shows binding characterization data for FluoSphere™-baseddetectable probes.

FIG. 44A shows stability characterization data for FluoSphere™-baseddetectable probes.

FIG. 44B shows stability characterization data for FluoSphere™-baseddetectable probes.

FIG. 44C shows stability characterization data for FluoSphere™-baseddetectable probes.

FIG. 45A shows binding characterization data for FluoSphere™-baseddetectable probes.

FIG. 45B shows binding characterization data for Alexa-Fluor®-basedaptamers.

FIG. 45C shows binding characterization data for APC-based aptamers.

FIG. 45D shows binding characterization data for SureLight™ APC-basedaptamers.

FIG. 46 shows binding data for FluoSphere™-based detectable probes withdiffering quantities of available affinity reagent attachment sites.

FIG. 47 shows binding data for fluorescent nanoparticle B1 probesfabricated with direct attachment or with pre-annealing protocol tohis-tagged Her2 (on target) and myoglobin (off-target).

FIG. 48 shows an immobilized target for selection of affinity reagents,along with an exemplary list of peptides which include the target.Figure discloses SEQ ID NOS 10-12, 1-2, 13, 3-4 and 14-17, respectively,in order of appearance.

FIG. 49A provides a schematic illustration of a labeled affinity reagentas described herein.

FIG. 49B shows a method of attaching label components to a nucleic acidprobe by enzymatic extension.

FIGS. 50A-50C diagram some exemplary ways to fluorescently label anaffinity reagent. FIG. 50A shows an aptamer with a single fluorophoreattached. FIG. 50B shows an aptamer with a double stranded nucleic acidlabel region, the label region having two fluorophores attached. FIG.50C shows attachment of multiple regularly spaced fluorophores to anaptamer using a template.

FIG. 51 shows a labeled probe including a single strand of nucleic acidincluding an aptamer, hybridized to a single strand of nucleic acidincluding fluorophores.

FIG. 52 shows a gel visualized at 488 nm to show double stranded DNA and647 nm to show incorporated fluorophores. These labeled affinityreagents were produced via enzymatic extension and incorporation offluorophore-modified nucleotides.

FIG. 53A shows a gel visualized at 488 nm to show double stranded DNA.

FIG. 53B shows a gel visualized at 647 nm to show incorporatedfluorophores. These labeled affinity reagents were produced viaenzymatic extension and incorporation of chemically-modifiednucleotides. Subsequently, chemical conjugation was used to incorporatedfluorophores, in accordance with some embodiments.

FIG. 54 shows labeled aptamer concentration.

FIG. 55A shows fluorophore concentration in the labeled aptamers of FIG.54 .

FIG. 55B shows a dUTP-647 standard curve used to determine thefluorophore concentration in FIG. 8A.

FIG. 56 shows the fluorophore:DNA ratio calculated from FIGS. 54 and 55.

FIG. 57A shows a labeled aptamer bound to an immobilized peptide.

FIG. 57B shows binding of a labeled aptamer to an immobilized targetpeptide.

FIG. 57C shows binding of a labeled aptamer to an immobilized non targetpeptide.

FIG. 57D shows microscopy of a labeled aptamer at limiting dilution onan amine-coated coverslip.

FIG. 58A shows a binding reaction between a detectable probe and aSNAP-attached polypeptide.

FIG. 58B shows a binding reaction between a plurality of detectableprobes and an array of SNAP-attached polypeptides.

DETAILED DESCRIPTION

Detection of interactions between affinity reagents and binding targetscan be useful for providing spatial and/or temporal characterizationand/or quantitation of physical systems. For example, affinityreagent-based methods such as enzyme linked immunosorbent assay (ELISA)may be used to determine the presence and potentially the quantity of abiomarker within a biochemical sample. Affinity reagents can displaycomplex behaviors, such as interactions with unexpected or unlikelytargets, or failure to interact with an expected target. In bulk orlarge-scale characterizations, affinity reagents may be engineered to asufficient degree that the complex aspects of their behavior fall withinthe experimental error, thereby producing usable interaction data.However, for single-molecule assays that utilize affinity reagents,complex affinity reagent behaviors may produce anomalous results (e.g.,““false”” negatives, false positives) that complicate the interpretationof single molecule affinity reagent interaction data.

Due to a variety of factors that influence binding interaction betweenan affinity reagent and a binding partner, binding may be transient. Insome cases, an affinity reagent may associate and disassociate freelywith a binding partner. In other cases, an affinity reagent may interactwith a binding partner with sufficient strength to create a stable orquasi-stable complex. Furthermore, in some cases an affinity reagent mayassociate with an unexpected or unlikely binding partner.

Due to the complex nature of the interactions between affinity reagentsand binding partners, some affinity reagent interactions may not fitwithin a conventional binary interaction framework (i.e., single bindingpartner, binding or no binding). For example, an affinity reagent maynot be observed interacting with an available binding partner forvarious reasons, including 1) failure of the affinity reagent to comewithin a sufficient proximity of the binding partner; 2) failure of theaffinity reagent to remain associated with the binding partner duringthe time interval when an observation of association is made; or 3)environmental changes that interrupt or weaken the affinity interaction(e.g., a conformational change of the binding partner or affinityreagent). Likewise, an affinity reagent may be observed binding to anunexpected or unlikely target for various reasons, such as 1)environmental changes that strengthen an affinity interaction; or 2) atransient association that occurs during the time interval when anobservation of association is made.

Consequently, some affinity reagent interactions may best be describedwithin a probabilistic or stochastic framework (e.g., multiple bindingpartners, non-zero probability of binding to each target). Probabilisticor stochastic models for affinity reagent binding may be especiallyuseful for single-molecule assays where single interaction measurementmay not necessarily provide a definitive characterization. In suchsituations, multiple rounds or cycles of measurement may increasemeasurement confidence.

Although affinity reagents may not always behave optimally in particularconditions, affinity reagents may be engineered to increase or improvetheir behavior for certain purposes. Two aspects of affinity reagentbehavior that may be modified include avidity and observability.

Avidity may be understood as the tendency of an affinity reagent toremain bound to a binding partner due to the presence of multiple, oftensynergistic, binding interactions between the affinity reagent andbinding partner. Multiple binding interactions can occur, for example,due to the binding partner having a plurality of different epitopes thatare recognized by the affinity reagent and/or due to the probe having aplurality of binding components that recognize an epitope in the bindingpartner. A common example of such a phenomenon is the avidity ofantibodies, where avidity is often achieved through the weak binding ofmultiple binding sites on a single antibody to a particular target tocreate an apparent binding strength that is greater than the bindingstrength between the binding partner and any individual binding site onthe antibody.

Observability may be understood as the ability to detect bindinginteraction between an affinity reagent and its binding partner. Forexample, observability may refer to the tendency of an affinity reagentto be detected during an interaction between the affinity reagent andits binding partner. In some configurations, observability may refer tothe tendency of an affinity reagent to create a signal or signalgenerating tag (e.g. a nucleic acid tag) that can be detected after aninteraction between the affinity reagent and its binding partner.Observability may be affected by both the ability of the affinityreagent to maintain an interaction for a sufficient time interval duringdetection and the resistance of the affinity to mechanisms that maydiminish a detectable signal of the interaction (e.g., photobleaching,cleavage of a label component).

Described herein are compositions including probes with enhanced avidityfor a binding partner. In some configurations, the probe is detectableand, optionally, has enhanced observability. Compositions describedherein, such as affinity reagents and detectable probes, can beespecially useful for single-molecule characterization assays including,for example, in configurations in which multiple rounds or cycles ofaffinity reagent interactions are measured. The compositions optionallyhave the property of enhanced avidity for one or more binding partnerswhile also being configured for reversible binding, for example, toallow removal from a binding partner to permit multiple cycles or roundsof binding measurements. The compositions can optionally possess tunabledetection labels that permit observation and measurement of the labelsat signal levels that sufficiently exceed the background signal of thesystem in which affinity agent interactions are measured.

In some configurations, detectable probes or affinity reagents describedherein may be characterized as including a retaining component that isassociated with one or both of a plurality of binding components and aplurality of label components (e.g. detection labels). The plurality ofbinding components may be displayed on the retaining component in aconfiguration that permits enhanced avidity for binding of thedetectable probe or affinity reagent to a particular binding partner.The affinity of the detectable probe or affinity reagent can exceed theaffinity of any one of the individual binding components for thatparticular binding partner. In some configurations, the avidity of thedetectable probe or affinity reagent for the binding partner may exceedthe sum of the affinities of the plurality of binding components for thebinding partner. Moreover, a plurality of label components may bedisplayed on the retaining component of a detectable probe or affinityreagent in a manner that enhances the detectable signal produced by thedetection labels. For example, signal can be enhanced with regard tointensity, duration or specificity. In particular configurations, adetectable probe or affinity reagent can further possess the property ofbeing stable in the presence of chemical species (e.g., surfactants,denaturants) that would otherwise disrupt interactions between anaffinity reagent and its binding partner, thereby permitting removal ofthe detectable probe or affinity reagent from the binding partnerwithout damaging or disrupting the structure of the detectable probe oraffinity reagent.

In some configurations, the described detectable probes or affinityreagents employ nucleic acid origami as a retaining component that iscoupled to a plurality of binding components (e.g. antibodies, antibodyfragments, mini proteins, DARPins, DNA aptamers, RNA aptamers, etc.)and/or a plurality of label components (e.g., fluorophores, nucleic acidtags, quantum dots, fluorescent nanoparticles, fluorescent proteins). Insome configurations, a nucleic acid origami-based retaining componentmay possess a regular or symmetric shape that permits attachment of aplurality of binding components and/or a plurality of label componentsat predetermined positions. For example, the components can be separatedfrom each other by predefined distances, the components can be orientedto achieve synergistic function, or the components can be oriented toreduce or prevent inhibition of each other's activity. The ability toadjust spacing and orientation can yield desired detection propertiessuch as reduced quenching between fluorophore labels or enhanced Forsterresonant energy transfer (FRET) between fluorophore labels.Alternatively or additionally, spacing and orientation can be adjustedto tune the affinity of the affinity reagent for one or more bindingpartner.

In particular configurations, a detectable probe can employ an opticallydetectable particle. The particle can have a modifiable surface to whicha plurality of binding components may be attached. The opticallydetectable particle may include a fluorescent or luminescentnanoparticle (e.g., a FluoSphere™, or quantum dot). The modifiablesurface coating may include a hydrogel or polymer. In suchconfigurations, the optically detectable particle may serve as both aretaining component for the attachment of binding components to thedetectable probe and as a label component for detection of the probe. Insome configurations, a surface coating of an optically detectableparticle may serve as a retaining component for the attachment ofbinding components to the detectable probe.

In further configurations, two or more detectable probes may be combinedto form a complex of detectable probes. A similar complex can be madebetween two affinity reagents or between an affinity reagent and adetectable probe. A complex of probes and/or reagents may possessbinding components of alike affinity (e.g., a univalent probe orreagent) or differing affinity (e.g., a plurivalent probe).

In some configurations, a detectable probe or affinity reagent iscombined with or coupled to a competitor affinity reagent. Thecompetitor affinity reagent may be configured as a free molecule, or asa binding component that is attached to the detectable probe or affinityreagent. Competitor affinity reagents have a decreased affinity orspecificity for a binding partner of interest in some cases resulting inhigher levels of promiscuity than a non-competitor affinity reagent. Thepresence of the competitor affinity reagent may enhance the avidity of adetectable probe or affinity reagent for a particular binding partner.

The present disclosure provides methods for detecting analytes usingcompositions including detectable probes or affinity reagents withenhanced avidity for a binding partner and enhanced observability.Methods are exemplified herein for use in detecting polypeptides. Itwill be understood that any of a variety of analytes can be used, suchas those that are targeted by analytical chemistry assays, biochemistryassays, molecular diagnostic assays, molecular forensic assays, qualitycontrol assays or the like.

Characterization and quantitation of heterogeneous polypeptide samplesis often hindered by the co-existence of proteins and/or peptides inwidely varying quantities. For example, the signal from a low-copynumber protein may be drowned out by the signal from a high-copy numberprotein in a quantitative characterization assay. Accuracy of apolypeptide characterization assay that is performed at a proteomiclevel (e.g. tens of thousands of unique protein species) can benefitfrom a combination of high-sensitivity analysis techniques andhigh-confidence prediction techniques.

Biological analyses can greatly benefit from advances in the toolsavailable for examining the molecular operations of biological systems.Advances in genomics technologies, single cell analysis platforms, andhighly sensitive chemical analysis systems can greatly improveresearcher's and clinician's view into how biological systems function,and in turn, improve understanding, treatment and prevention of diseaseand other human health conditions.

Many of these advanced tools benefit from the use of detectable reagentsthat are able to bind, or otherwise associate with different biologicalmolecules, where the binding allows for the identification of thepresence and/or the quantification of such molecules in a given system,where that presence and/or quantity provides insight into thefunctioning of the system. As an example, many of these tools providefor the immobilization of different molecular species from a biologicalsystem, such as proteins, nucleic acids or the like, on a supportingstructure, such as a fixed substrate or bead. Molecules that have alevel of binding affinity for different molecular species, also calledaffinity reagents, may then be used to interrogate the bound moleculesby contacting the two together to see if, where, and/or for how long theaffinity reagents bind, indicating the potential presence of particularmolecules in the biological sample.

Detection of binding can typically be accomplished through the detectionof a label component of the affinity reagent. While the label componentmay be an inherent part of the affinity reagent, in many cases, thelabel component may be an exogenous chemical or structural moietyattached to the affinity reagent. An affinity reagent having a labelcomponent can function as a detectable probe.

A wide variety of exogenous detectable labels may generally be usefulfor such purposes, including, for example, optically detectable labels,such as fluorescent dye labels, enzymes (for example enzymes whichcatalyze reactions with colored reagents or products), electrochemicallabels, such as highly charged label groups, or even groups that aredetected through subsequent processing, such as nucleic acid barcodelabels that can be subsequently identified through nucleic acidamplification processes, sequencing processes or hybridization assays.Fluorescent labels are useful for visualization of binding interactionsbetween affinity reagents and other molecules, such as proteins orpeptides.

Depending on the applications for which affinity reagents or detectableprobes will be used, different degrees of signal intensity may bedesirable, or even required. By way of example, in many applicationsamplification of a binding signal may be achieved through the binding ofmany affinity reagents or detectable probes. For example a sample may beseparated such that multiple molecules of a single detectable moiety areaggregated in a single location, and are thus, more readily observed ordetected. Alternatively or additionally, secondary binding systems maybe used to present multiple additional binding sites to which separatelabel components may be subsequently bound and detected.

In some cases, however, it may be useful to visualize binding of asingle molecule of an affinity reagent or detectable probe to a singlemolecule in a sample in a quantitative manner. As will be appreciated,detection of individual molecules presents many challenges in terms ofdetection. One such challenge is the ability to present a detectablesignal from the binding of a single molecule of reagent or probe to asingle molecule that has sufficient signal strength (e.g. signalintensity and duration) as to be detected by available analyticalsystems, both in terms of raw signal strength, as well as signalstrength relative to strength of background noise of the system.

The signal strength challenge may be addressed through a number ofapproaches, alone or in combination. For example, use of highlysensitive microscopy techniques can enhance abilities to detect very lowlevel signals, including single molecule signals having low intensityand/or short duration. Additionally, signal strength may be increasedthrough the incorporation of multiple label components (e.g., multiplelabel components on a single molecule such as an affinity reagentmolecule or detectable probe molecule) to significantly increase thesignal associated with that single molecule, and thus improve itsdetectability. Again, by way of example, for fluorescent labels,increasing signal intensity may be accomplished by attaching severalfluorescent moieties in a single affinity reagent or detectable probe.However, simply loading up a probe or reagent with fluorescent dyelabels can raise its own set of challenges. Care may be taken toconfigure a probe or reagent such that one fluorescent moiety on theprobe or reagent does not quench another fluorescent moiety on the probeor reagent, that the number of fluorescent moieties on a probe orreagent can be controlled, and that the chemistry of attaching theselabeling groups to the probe or reagent does not interfere with thebinding affinity of the probes themselves.

Particularly useful affinity reagents and detectable probes are able tobind to specific proteins, metabolites, cells or cell interfaces.Examples of such affinity reagents and detectable probes may includeprotein-based probes composed of naturally occurring amino acids, smallmolecule probes, nucleic acid-based probes composed of naturallyoccurring bases, and probes composed of non-natural nucleotides andamino acids. The reagents can be configured to bind exclusively to agiven epitope or other target.

Exclusivity of binding is often considered to be a desirable trait in anaffinity reagent or detectable probe. Substantial efforts are made toensure that the affinity reagent or probe binds to just one targetspecies, with minimal binding to other targets. For example, the targetspecies may be a particular sequence of amino acids. If that sequence isunique to a protein that is found in a biological sample, then a reagentor probe that is specific for the sequence will be specific for theprotein in the milieu of the sample. As such, the reagent or probe canbe used to identify or even quantify the protein in the sample. As setforth in further detail herein, there are particular use cases in whichit may be useful to have affinity reagents or detectable probes thatbind to one or more different proteins in a sample. For example, auseful affinity reagent or detectable probe may bind to an epitope thatis common to two or more different proteins. Alternatively oradditionally, the probe or reagent may bind to two or more differentepitopes, the epitopes being present in different proteins or indifferent regions of the same protein. Affinity probes and detectableprobes that bind to multiple targets in a sample can be employed touseful effect in compositions and methods set forth herein.

Terms used herein will be understood to take on their ordinary meaningin the relevant art unless specified otherwise. Several terms usedherein and their meanings are set forth below.

As used herein, the term “affinity reagent” refers to a molecule orother substance that is capable of binding to a binding partnerspecifically, reproducibly or with high probability. Specific bindingcan be characterized in terms of a binding constant such as dissociationconstant (K_(D)) that is less 10⁻⁴ M, 10⁻⁶ M, 10⁻⁸ M, 10⁻¹⁰ M, 10⁻¹² M,10⁻¹⁴ M or lower. High probability can be characterized as a probabilitythat is at least 0.25, 0.5, 0.51, 0.75, 0.9, 0.99 or higher (on a scaleof 0 to 1). An affinity reagent can optionally be larger than, smallerthan or the same size as its binding partner. An affinity reagent mayform a reversible or irreversible interaction with a binding partner. Anaffinity reagent may bind with a binding partner in a covalent ornon-covalent manner. An affinity reagent is typically non-catalytic andchemically non-reactive, thereby not permanently altering the chemicalstructure of the binding partner it binds in a method set forth herein.Alternatively, an affinity reagent may be configured to catalyze orparticipate in a chemical modification (e.g., ligation, cleavage,concatenation, etc.) that produces a detectable change in a bindingpartner to which it binds. Optionally, the product of the reaction canpermit detection of the interaction. Affinity reagents may includereactive affinity reagents (e.g., kinases, ligases, proteases,nucleases, etc.) or non-reactive affinity reagents (e.g., antibodies,antibody fragments, aptamers, DARPins, peptamers, etc.). An affinityreagent may include one or more known and/or characterized bindingcomponents or binding sites (e.g., complementarity-defining regions)that mediate or facilitate binding with a binding partner. Accordingly,an affinity reagent can be monovalent (e.g. having only a single bindingcomponent), bivalent (e.g. having only two binding components),trivalent (e.g. having only three binding components), tetravalent (e.g.having only four binding components) or multivalent (e.g. having two ormore binding components). Exemplary affinity reagents include detectableprobes and probes as set forth in U.S. Provisional Application No.63/112,607, which is incorporated herein by reference.

As used herein, the term “antibody” refers to an immunoglobulinmolecule, or functional fragment thereof that specifically binds abinding partner. An antibody may have specificity for one or moreepitopes within a binding partner. An antibody may benaturally-occurring, engineered, or evolved. An antibody may includecomplementarity determining region (CDR) fragments, single-chainantibodies (scFv), single domain antibodies, chimeric antibodies,diabodies, and polypeptides that contain at least a portion of animmunoglobulin that is sufficient to confer specific epitope binding tothe polypeptide. Linear antibodies are also included for the purposesdescribed herein. Exemplary antibodies include immunoglobulin isotypessuch as IgM, IgA, IgG, IgD, and IgE. Exemplary antibody fragmentsinclude F(ab′)2 fragments, Fab′ fragments, Fab fragments, Fv fragments,scFV fragments, r1gG fragments, and Fc fragments.

As used herein, the term “approximately,” when used in connection withshapes, may mean a shape that is within 20% of an ideal shape withreference to two or more measures of the shape. For example, FIGS. 2A-2Bshow an approximately square and approximately circular 2-dimensionalbodies, 210 and 215 respectively, with ideal boundaries for the square220 or circle 225 shown in dashed lines. As used herein, the term“approximate,” when used in connection with a length, area, or volumemay mean a length, area, or volume that is within 10% of the givenmeasurement. For example, a length of 10 millimeters (mm) may refer toany length between 9 mm and 11 mm.

As used herein, the term “array” refers to a population of analytes(e.g. proteins) that are associated with unique identifiers such thatthe analytes can be distinguished from each other. A unique identifiercan be a solid support (e.g. particle or bead), structured nucleic acidparticle, retaining component, site (e.g. spatial address) on a solidsupport, tag, label (e.g. luminophore), or barcode (e.g. nucleic acidbarcode) that is associated with an analyte and that is distinct fromother identifiers in the array. Analytes can be associated with uniqueidentifiers by attachment, for example, via covalent or non-covalent(e.g. ionic bond, hydrogen bond, van der Waals forces, electrostaticsetc.) bonds. An array can include different analytes that are eachattached to different unique identifiers. An array can include differentunique identifiers that are attached to the same or similar analytes. Anarray can include separate solid supports or separate addresses thateach bear a different analyte, wherein the different analytes can beidentified according to the locations of the solid supports oraddresses. Analytes or other molecules that can be included in an arraycan be, for example, nucleic acids such as SNAPs, polypeptides, enzymes,affinity reagents, biding partners, ligands, or receptors.

As used herein, the term “attached” refers to the state of two thingsbeing joined, fastened, adhered, connected or bound to each other. Forexample, a binding component can be attached to a retaining component bya covalent or non-covalent bond. A covalent bond is characterized by thesharing of pairs of electrons between atoms. A non-covalent bond is achemical bond that does not involve the sharing of pairs of electronsand can include, for example, hydrogen bonds, ionic bonds, van der Waalsforces, hydrophilic interactions and hydrophobic interactions.

As used herein, the term “binding affinity” or “affinity” refers to thestrength or extent of binding between an affinity reagent and a bindingpartner, epitope or target moiety. In some cases, the binding affinityof an affinity reagent for a binding partner, epitope, or target moietymay be vanishingly small or effectively zero. A binding affinity of anaffinity reagent—of an affinity reagent for a binding partner, epitope,or target moiety may be qualified as being a “high affinity,” “mediumaffinity,” or “low affinity.” A binding affinity—of an affinity reagentfor a binding partner, epitope, or target moiety may be quantified asbeing “high affinity” if the interaction has an equilibrium dissociationconstant ((K_(D)) of less than about 100 nM, “medium affinity” if theinteraction has a dissociation constant between about 100 nM and 1 mM,and “low affinity” if the interaction has a dissociation constant ofgreater than about 1 mM. Binding affinity—can be described in termsknown in the art of biochemistry such as equilibrium dissociationconstant, equilibrium association constant (K_(A)), association rateconstant (k_(on)), dissociation rate constant (k_(off)) and the like.See, for example, Segel, Enzyme Kinetics John Wiley and Sons, New York(1975), which is incorporated herein by reference in its entirety.

As used herein, the term “binding component” refers to a moiety of anaffinity reagent, the moiety being capable of binding to a bindingpartner specifically, reproducibly or with high probability. Exemplarybinding components include, but are not limited to, antibodies orfunctional fragments thereof (e.g., Fab′ fragments, F(ab′)₂ fragments,single-chain variable fragments (scFv), di-scFv, tri-scFv,microantibodies, intrabodies, affibodies, affilins, affimers, affitins,alphabodies, anticalins, avimers, DARPins, Kunitz domain peptides,monobodies, nanoCLAMPs, mini-peptide binders, etc.), lectins orfunctional fragments thereof, avidin, streptavidin, aptamers, nucleicacids that are either single- or double-stranded, or other affinityreagents set forth herein or known in the art. Exemplary bindingcomponents include probes, as set forth in U.S. Provisional ApplicationNo. 63/112,607, which is incorporated herein by reference.

As used herein, the term “binding context” refers to the environmentalconditions in which an affinity reagent-binding partner interaction isobserved. Environmental conditions may include any factors that mayinfluence an interaction between an affinity reagent and a bindingpartner, such as temperature, fluid properties (e.g., ionic strength,pH), relative concentrations, absolute concentrations, fluidcomposition, binding partner conformation, affinity reagentconformation, and combinations thereof. Environmental conditions mayinclude structural features of a binding partner that, although beingoutside of an epitope, have an influence interaction of the epitope witha binding reagent. The structural features can include, for example,amino acids or regions of a polypeptide that are proximal to an epitopein the primary, secondary, tertiary or quaternary structure of thepolypeptide.

As used herein, the term “binding partner” refers to molecule or othersubstance that is recognized by an affinity reagent or bindingcomponent. An affinity reagent may specifically or reproduciblyrecognize a particular binding partner relative to other molecules orsubstances in a sample. Alternatively, an affinity reagent may recognizea plurality of different binding partners in a sample, for example,binding promiscuously to a subset of different polypeptide sequencesamong a larger set of different polypeptides. A binding partner may becapable of forming an interaction with an affinity reagent, regardlessof whether such an interaction occurs. A binding partner may include oneor more epitopes. A binding partner may have a rigid structure, such asa nanoparticle or a microparticle. A binding partner may have a moltenor dynamic structure (e.g., a globular protein, a globular polymer). Abinding partner may be solution phase or solid phase. For example abinding partner can be free within a solution containing an affinityreagent, or may be localized at a surface or interface that an affinityreagent can access. A binding partner can be attached to a structurednucleic acid particle (e.g. nucleic acid origami) or retainingcomponent.

As used herein, the term “binding probability” refers to the probabilitythat an affinity reagent may be observed to interact with a bindingpartner and/or an epitope, for example, within a fixed binding context.A binding probability may be expressed as a discrete number (e.g., 0.4or 40%) a matrix of discrete numbers, or as mathematical model (e.g., atheoretical or empirical model). A binding probability may include oneor more factors, including binding specificity, likelihood of locating atarget epitope, or the likelihood of binding for a sufficient amount oftime for a binding interaction to be detected. An overall bindingprobability may include binding probability when all factors have beenweighted relative to the binding context.

As used herein, the term “binding specificity” refers to the tendency ofan affinity reagent to preferentially interact with a binding partner orepitope relative to other biding partners or epitopes. An affinityreagent may have a calculated, observed, known, or predicted bindingspecificity for any possible binding partner or epitope. Bindingspecificity may refer to selectivity for a single binding partner,epitope, or target moiety in a sample over all, some or at least oneother analyte in the sample. Moreover, binding specificity may refer toselectivity for a subset of binding partners, epitopes, or targetmoieties in a sample over at least one other analyte in the sample.

As used herein, the term “bioorthogonal reaction” refers to a chemicalreaction that can occur within a biological system (in vitro and/or invivo) without interfering with some or all native biological processes,functions, or activities of the biological system. A bioorthogonalreaction may be further characterized as being inert to components of abiological system other than those targeted by the bioorthogonalreaction. A bioorthogonal reaction may include a click reaction.Bioorthogonal or click reactions may include Staudinger ligation,copper-free click reactions, nitrone dipole cycloaddition, norbornenecycloaddition, oxanobornadiene cycloaddition, tetrazine ligation, [4+1]cycloaddition, tetrazole photoclick reactions, or quadricyclaneligation. A bioorthogonal reaction may utilize an enzymatic approach,such as attachment between a first molecule and a second molecule by anenzyme such as a sortase, a ligase, or a subtiligase. A bioorthogonalreaction may utilize an irreversible peptide capture system, such asSpyCatcher/SpyTag, SnoopCatcher/SnoopTag, or SdyCatcher/SdyTag.

As used herein, the term “click reaction” refers to single-step,thermodynamically-favorable conjugation reaction utilizing biocompatiblereagents. A click reaction may be configured to not utilize toxic orbiologically incompatible reagents (e.g., acids, bases, heavy metals) orto not generate toxic or biologically incompatible byproducts. A clickreaction may utilize an aqueous solvent or buffer (e.g., phosphatebuffer solution, Tris buffer, saline buffer, MOPS, etc.). A clickreaction may be thermodynamically favorable if it has a negative Gibbsfree energy of reaction, for example a Gibbs free energy of reaction ofless than about −5 kiloJoules/mole (kJ/mol), −10 kJ/mol, −25 kJ/mol, −50kJ/mol, −100 kJ/mol, −200 kJ/mol, −300 kJ/mol, −400 kJ/mol, or less than−500 kJ/mol. Exemplary bioorthogonal and click reactions are describedin detail in WO2019/195633A1, which is incorporated herein by reference.

The term “comprising” is intended herein to be open-ended, including notonly the recited elements, but further encompassing any additionalelements.

As used herein, the term “each,” when used in reference to a collectionof items, is intended to identify an individual item in the collectionbut does not necessarily refer to every item in the collection.Exceptions can occur if explicit disclosure or context clearly dictatesotherwise.

As used herein, the term “epitope” refers to a molecule or part of amolecule, which is recognized by or binds specifically to an affinityreagent. Epitopes may include amino acid sequences that are sequentiallyadjacent in the primary structure of a polypeptide or amino acids thatare structurally adjacent in the secondary, tertiary or quaternarystructure of a polypeptide. An epitope can optionally be recognized byor bound to an antibody. However, an epitope need not necessarily berecognized by any antibody, for example, instead being recognized by anaptamer, miniprotein or other affinity agent. An epitope can optionallybind an antibody to elicit an immune response. However, an epitope neednot necessarily participate in, nor be capable of, eliciting an immuneresponse. The term “affinity target” is used herein synonymously withthe term “epitope.”

As used herein, the term “exogenous,” when used in reference to a moietyof a molecule, means a moiety that is not present in a natural analog ofthe molecule. For example, an exogenous label of an amino acid is alabel that is not present on a naturally occurring amino acid.Similarly, an exogenous label that is present on an antibody is notfound on the antibody in its native milieu.

As used herein, the term “functional group” refers to a moiety or groupof atoms in a molecule that confer a chemical property, such asreactivity, polarity, hydrophobicity, hydrophilicity, solubility,binding affinity etc., to the molecule. Functional groups may includeorganic moieties or may include inorganic atoms. Exemplary functionalgroups may include bioorthogonal reactants, click reactants, alkyl,alkenyl, alkynyl, phenyl, halide, hydroxyl, carbonyl, aldehyde, acylhalide, ester, carboxylate, carboxyl, carboalkoxy, methoxy, hydroperoxy,ether, hemiacetal, hemiketal, acetal, ketal, orthoester, epoxide,carboxylic anhydride, carboxamide, amine, ketimine, aldimine, imide,azide, azo, cyanate, isocyanate, nitrate, nitrile, isonitrile,nitrosoxy, nitro, nitroso, oxime, pyridyl, carbamate, sulfhydryl,sulfide, disulfide, sulfinyl, sulfonyl, sulfinom, sulfo, thiocyanate,isothiocyanate, carbonothioyl, thioester, thionoester, phosphino,phosphono, phosphonate, phosphate, borono, boronate, and borinatefunctional groups.

As used herein, the term “label” refers to a molecule, or moietythereof, that provides a detectable characteristic. The detectablecharacteristic can be, for example, an optical signal such as absorbanceof radiation, luminescence emission, luminescence lifetime, luminescencepolarization, fluorescence emission, fluorescence lifetime, fluorescencepolarization, or the like; Rayleigh and/or Mie scattering; bindingaffinity for a ligand or receptor; magnetic properties; electricalproperties; charge; mass; radioactivity or the like. Exemplary labelsinclude, without limitation, a fluorophore, luminophore, chromophore,nanoparticle (e.g., gold, silver, carbon nanotubes), heavy atoms,radioactive isotope, mass label, charge label, spin label, receptor,ligand, or the like. A label may produce a signal that is detected inreal-time (e.g., fluorescence, luminescence, radioactivity). A labelcomponent may produce a signal that is detected off-line (e.g., anucleic acid barcode) or in a time-resolved manner (e.g., time-resolvedfluorescence). A label component may produce a signal with acharacteristic frequency, intensity, polarity, duration, wavelength,sequence, or fingerprint.

As used herein, the term “label component” refers to a moiety of anaffinity reagent or other substance that provides a detectablecharacteristic. The detectable characteristic can be, for example, anyof those set forth herein in the context of labels. A label componentcan be attached to or capable of being attached to another molecule orsubstance. Exemplary molecules that can be attached to a label componentinclude an affinity reagent or a binding partner.

As used herein, the term “nucleic acid nanoball” refers to a globular orspherical nucleic acid structure. A nucleic acid nanoball may include aconcatemer of sequence regions that arranges in a globular structure. Anucleic acid nanoball may include DNA, RNA, PNA, modified or non-naturalnucleic acids, or combinations thereof.

As used herein, the term “nucleic acid origami” refers to a nucleic acidconstruct including an engineered tertiary or quaternary structures inaddition to the naturally-occurring helical structure of nucleicacid(s). A nucleic acid origami may include DNA, RNA, PNA, modified ornon-natural nucleic acids, or combinations thereof. A nucleic acidorigami may include a plurality of oligonucleotides that hybridize viasequence complementarity to produce the engineered structuring of theorigami particle. A nucleic acid origami may include sections ofsingle-stranded or double-stranded nucleic acid, or combinationsthereof. Exemplary nucleic acid origami structures may includenanotubes, nanowires, cages, tiles, nanospheres, blocks, andcombinations thereof. A nucleic acid origami can optionally include arelatively long scaffold nucleic acid to which multiple smaller nucleicacids hybridize, thereby creating folds and bends in the scaffold thatproduce an engineered structure. The scaffold nucleic acid can becircular or linear. The scaffold nucleic acid can be single stranded butfor hybridization to the smaller nucleic acids. A smaller nucleic acid(sometimes referred to as a “staple”) can hybridize to two regions ofthe scaffold, wherein the two regions of the scaffold are separated byan intervening region that does not hybridize to the smaller nucleicacid.

As used herein, the term “oligonucleotide” refers to a moleculeincluding two or more nucleotides joined by a phosphodiester bond. Anoligonucleotide may include DNA, RNA, PNA, modified nucleotides,non-natural nucleotides, or combinations thereof. An oligonucleotide mayinclude a limited number of nucleotide subunits, such as, for example,less than about 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100,90, 80, 70, 60, 50, 40, 30, 25, 20, 15, 10, or less than 5 nucleotides.

As used herein, the term “offset,” when used in reference to molecularstructures, refers to the spatial difference in orientation between twolines (2-dimensional) or two planes (3-dimensional). The planes canapproximate a surface of a molecular structure such as a face of anorigami tile. An offset may include a distance offset and/or an angularoffset. FIGS. 1A and 1B depict examples of angular offset for differingtwo-dimensional shapes (which could be two-dimensional projections ofthree-dimensional structures). The isosceles triangle 100 of FIG. 1A hasan angular offset of 120° between the first face 110 and the second face120 whose relative orientations are depicted by orthogonal vectors A andA′. The rectangle 130 of FIG. 1B has an angular offset of 180° betweenthe first face 110 and the second face 120, whose relative orientationsare depicted by orthogonal vectors A and A′.

As used herein, the term “polypeptide” refers to a molecule includingtwo or more amino acids joined by a peptide bond. A polypeptide may alsobe referred to as a protein, oligopeptide or peptide. Although the terms“protein,” “polypeptide,” “oligopeptide” and “peptide” may optionally beused to refer to molecules having different characteristics, such asamino acid sequence composition or length, molecular weight, origin ofthe molecule or the like, the terms are not intended to inherentlyinclude such distinctions in all contexts. A polypeptide can be anaturally occurring molecule, or synthetic molecule. A polypeptide mayinclude one or more non-natural amino acids, modified amino acids, ornon-amino acid linkers. A polypeptide may contain D-amino acidenantiomers, L-amino acid enantiomers or both. Amino acids of apolypeptide may be modified naturally or synthetically, such as bypost-translational modifications.

As used herein, the term “promiscuity,” when used in reference to anaffinity reagent, refers to the binding agent binding to, or having thecapability of binding to, two or more different binding partners. Forexample, a promiscuous binding agent may: 1) bind to a plurality ofdifferent binding partners due to the presence of a common epitopewithin the structures of the different binding partner; or 2) bind to aplurality of different epitopes; or 3) a combination of both properties.A promiscuous binding agent may bind to a plurality of binding partnersdue to the presence of a particular epitope or target moiety, regardlessof the binding context of the epitope or target moiety. The bindingcontext may include, for example, the local chemical environmentsurrounding an epitope or target moiety, such as flanking, adjacent, orneighboring chemical entities (e.g., for a polypeptide epitope, flankingamino acid sequences, or adjacent or neighboring non-contiguous aminoacid sequences relative to the epitope). A plurality of differentepitopes that is bound by a promiscuous affinity reagent may includestructurally- or chemically-related epitopes that nonetheless havedifferent amino acid content. For example, an affinity reagent may beconsidered promiscuous if it possesses a binding affinity for trimerpeptide sequences having the form WXK, where X is any possible aminoacid. Additional concepts pertaining to binding promiscuity arediscussed in WO 2020/106889A1, which is incorporated herein byreference.

As used herein, the term “retaining component” refers to a moiety of anaffinity reagent, detectable robe or other substance that links at leasttwo other components. A retaining component can maintain two othercomponents within a particular distance of each other. For example, thetwo other components can be maintained at a distance of at most 1000 nm,500 nm, 100 nm, 50 nm, 10 nm, 5 nm, 1 nm or less. Alternatively oradditionally, a retaining component can separate the two other moietiesat a minimum distance from each other. For example, the two othercomponents can be maintained at a distance of at least 1 nm, 5 nm, 10nm, 50 nm, 100 nm, 500 nm, 1000 nm or more. A retaining component caninclude, for example, a structured nucleic acid particle, nucleic acidnanoball, nucleic acid origami, protein nucleic acid, polypeptide,synthetic polymer, polysaccharide, organic particle, inorganic particle,gel, hydrogel, coated particle, or the like. A retaining component canoptionally have a polymeric structure. Alternatively, a retainingcomponent need not have a polymeric structure. In some embodiments, aretaining component has a composition that is similar to othercomponents to which it is attached. For example, a plurality of bindingcomponents that are composed of polypeptide material can be attached toa polypeptide retaining component. Alternatively, a retaining componentcan have a composition that differs substantially from the compositionof other components to which it is attached. For example, a plurality ofbinding components that are composed of polypeptide material can beattached to a retaining component that is composed partially or entirelyof a material other than polypeptide, such as nucleic acid material, oran organic or inorganic nanoparticle (e.g., carbon nanosphere, silicondioxide nanosphere, etc.). A retaining component may include one or moreattachment sites that permit attachment of another component, such as alabel component or binding component, to the retaining component.Attachment sites may include functional groups, active sites, bindingligands, binding receptors, nucleic acid sequences, or any other entitycapable of forming a covalent or non-covalent attachment to a bindingcomponent, label component, or other detectable probe component. Aretaining component may include an organic or inorganic particle ornanoparticle. A scaffold may include a coating or surface layer thatpermits attachment of another component, for example, as occurs in apolymer-coated FluoSphere™ or polymer-coated quantum dot. Examples ofretaining components include scaffolds as set forth in U.S. ProvisionalApplication No. 63/112,607, which is incorporated herein by reference.

As used herein, the term “site,” when used in reference to an array,means a location in an array occupied by, or configured to be occupiedby, a particular molecule or analyte such as a polypeptide, nucleicacid, structured nucleic acid or functional group. A site can containonly a single molecule, or it can contain a population of severalmolecules of the same species (i.e. an ensemble of the molecules).Alternatively, a site can include a population of molecules that aredifferent species. Sites of an array are typically discrete. Thediscrete sites can be contiguous, or they can have interstitial spacesbetween each other. An array useful herein can have, for example, sitesthat are separated by less than 100 microns, 10 microns, 1 micron, or0.5 micron, 0.1 micron, 0.01 micron or less. Alternatively oradditionally, an array can have sites that are separated by at least0.01 micron, 0.1 micron, 0.5 micron, 1 micron, 10 microns, 100 micronsor more. The sites can each have an area of less than 1 squaremillimeter, 500 square microns, 100 square microns, 25 square microns, 1square micron or less. An array can include at least about 1×10⁴, 1×10⁵,1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², or more sites.

As used herein, the term “solid support” refers to a rigid substratethat is insoluble in aqueous liquid. The substrate can be non-porous orporous. The substrate can optionally be capable of taking up a liquid(e.g. due to porosity) but will typically be sufficiently rigid that thesubstrate does not swell substantially when taking up the liquid anddoes not contract substantially when the liquid is removed by drying. Anonporous solid support is generally impermeable to liquids or gases.Exemplary solid supports include, but are not limited to, glass andmodified or functionalized glass, plastics (including acrylics,polystyrene and copolymers of styrene and other materials,polypropylene, polyethylene, polybutylene, polyurethanes, Teflon™,cyclic olefins, polyimides etc.), nylon, ceramics, resins, Zeonor™,silica or silica-based materials including silicon and modified silicon,carbon, metals, inorganic glasses, optical fiber bundles, and polymers.

As used herein, the term “species” is used to identify molecules ormoieties that share the same chemical structure. For example, individualepitope moieties that have the same sequence of amino acids are the samespecies of epitope, whereas epitope moieties with different sequencesare different species of epitope. Proteins expressed from the same geneare the same species of gene product, proteins expressed from the samegene and having the same post-translational modifications are the sameproteoform or isoform. Two proteins can be expressed from the same genebut have different protein modifications, in which case the two proteinsare the same species of gene product but different proteoforms orisoforms.

As used herein, the term “structured nucleic acid particle” (or “SNAP”)refers to a single- or multi-chain polynucleotide molecule having acompacted three-dimensional structure. The compacted three-dimensionalstructure can optionally be characterized in terms of hydrodynamicradius or Stoke's radius of the SNAP relative to a random coil or othernon-structured state for a nucleic acid having the same sequence lengthas the SNAP. The compacted three-dimensional structure can optionally becharacterized with regard to tertiary structure. For example, a SNAP canbe configured to have an increased number of internal bindinginteractions between regions of a polynucleotide strand, less distancebetween the regions, increased number of bends in the strand, and/ormore acute bends in the strand, as compared to the same nucleic acidmolecule in a random coil or other non-structured state. Alternativelyor additionally, the compacted three-dimensional structure canoptionally be characterized with regard to quaternary structure. Forexample, a SNAP can be configured to have an increased number ofinteractions between polynucleotide strands or less distance between thestrands, as compared to the same nucleic acid molecule in a random coilor other non-structured state. In some configurations, the secondarystructure (i.e. the helical twist or direction of the polynucleotidestrand) of a SNAP can be configured to be more dense than the samenucleic acid molecule in a random coil or other non-structured state. ASNAP can optionally be modified to permit attachment of additionalmolecules to the SNAP. A SNAP may comprise DNA, RNA, PNA, modified ornon-natural nucleic acids, or combinations thereof. A SNAP may include aplurality of oligonucleotides that hybridize to form the SNAP structure.The plurality of oligonucleotides in a SNAP may include oligonucleotidesthat are attached to other molecules (e.g., affinity reagents, bindingpartners, functional groups, or detectable labels) or are configured tobe attached to other molecules (e.g., by functional groups). A SNAP mayinclude engineered or rationally-designed structures, such as nucleicacid origami.

As used herein, the term “substantially the same,” when used inreference to two or more structures, refers to the structuresperforming, or being capable of performing, substantially the samefunction in substantially the same way to obtain the same result. Forexample, two structured nucleic acid particles that are substantiallythe same can have the same primary structure. Optionally, they maydiffer in primary structure so long as they do not differ in tertiary orquaternary structure. Optionally, they may differ in one or more ofprimary, secondary, tertiary or quaternary structure, so long as theyperform substantially the same function in substantially the same way toobtain the same result in a method set forth herein.

As used herein, the term “target moiety” refers to a specific chemicalstructure within an epitope that mediates or facilitates a bindinginteraction. A target moiety may include a functional group, sidechain,active site, or other chemical entity with a characterizable structure.For example, a target moiety can be one or more amino acids, orsidechain(s) thereof, that form a portion of a polypeptide epitope. Atarget moiety may specifically interact with a binding site of anaffinity reagent to facilitate or mediate the interaction that causesbinding of the affinity reagent to the binding partner.

As used herein the term “tunable” refers to adjustability of thespecific, precise, and/or rational location of components or attachmentsites in the structure of a retaining component, scaffold or molecule.Tunable retaining components may refer to the ability to attach othercomponents at specific sites or within specific regions of the retainingcomponent structure, or to generate attachment sites for the attachingof other components at specific sites or specific regions of theretaining component structure. As used herein, “tunability” refers tothe property of a probe or retaining component having a tunablestructure or architecture.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” As used herein, the term “about,” when used in connection withpercentages, may mean±5% of the value being referred to. For example,about 90% would mean from 85% to 95%.

As used herein, the term “two-dimensional projection” refers to the areaor shape that would be occupied by the projection of a three-dimensionalstructure onto a planar two-dimensional surface without substantialgeometric or spatial distortion. For example, the two-dimensionalprojection of a sphere onto a planar two-dimensional surface wouldproduce a circular area on the surface with a diameter equivalent to thediameter of the sphere. A two-dimensional projection may be formed fromany frame of reference, including a frame of reference that isorthogonal to any surface of the three-dimensional structure.

Structure and Function of Detectable Probes and Affinity Reagents

Described herein are detectable probes or affinity reagents having anincreased binding avidity for a binding partner, increased observabilityor both. Structural and functional characteristics exemplified hereinfor detectable probes can be present in other affinity reagents.Conversely, structural and functional characteristics exemplified hereinfor affinity reagents can be present in detectable probes. Accordingly,the compositions, structures and methods set forth herein in the contextof detectable probes can be applied to other affinity reagents and viceversa.

Detectable probes or affinity reagents may include a plurality ofbinding components that, collectively, increase the overall bindingaffinity of the probe for a binding partner as compared to anyindividual binding component of the plurality of binding componentscontained within the detectable probe or affinity reagent structure. Anyof a variety of affinity reagents known in the art or set forth hereincan function as binding components when attached to a detectable probeor affinity reagent of the present disclosure. Detectable probes oraffinity reagents of the present disclosure may further include one ormore label components that permit observation of a binding interactionbetween the detectable probe (or affinity reagent) and a bindingpartner. Any of a variety of detectable labels known in the art or setforth herein can function as label components when attached to adetectable probe or affinity reagent of the present disclosure.

A detectable probe or affinity reagent of the present disclosure mayinclude (a) a retaining component; (b) a label component, and (c) one,two or more binding components attached to the retaining component. Ofparticular interest are retaining components that are spatially and/ororientationally tunable to provide precise, predetermined and/orrational display of other components attached thereto. For example,binding components can be attached in configurations that increase thebinding avidity and/or observability of the detectable probe or affinityreagent. A detectable probe or affinity reagent may include a retainingcomponent that provides sufficient sites to attach at least one bindingcomponent and/or at least one label component. In some configurations,the retaining component may be a natural, artificial, or syntheticparticle including a plurality of functional groups, reactive sites,nucleic acids, or functional groups that permit attachment of othermolecules to the retaining component. A retaining component may have anamorphous, globular, or irregular structure (e.g., a DNA nanoball, afluorescent nanoparticle such as a FluoSphere™ or a quantum dot). Aretaining component may have a regular or symmetric structure (e.g.,carbon nanospheres, carbon nanotubes, metal nanotubes, ceramicnanoparticles). A retaining component may include a shell or scaffoldthat is formed by a templating particle, such as a particle ornanoparticle. In some configurations, a templating particle may includea label component, such as a FluoSphere™ or a quantum dot. A retainingcomponent may include any appropriate material, including, but notlimited to polymers, metals, semiconductors, ceramics, glasses, andbiomolecules (e.g., nucleic acids such as DNA or RNA, proteins,polysaccharides).

In some cases, a retaining component may include a partially orsubstantially completely double stranded nucleic acid. A double strandednucleic acid may impart additional structural rigidity to the retainingcomponent, in order to provide better spacing between label components.In such cases, the label components may be coupled to one or bothstrands of the double stranded nucleic acid. A retaining component mayinclude a structured particle or rationally-designed particle. Aretaining component may include a structured nucleic acid particle(SNAP) that is configured to attach one or more binding componentsand/or one or more label components. In some configurations, the SNAPmay include a DNA origami particle or a DNA nanoball.

A retaining component may include a plurality of sites for attachingother components (e.g. a binding component, label component or anotherretaining component). A retaining component may include unique ordedicated sites for coupling binding components and/or label components.For example, a retaining component may include nucleic acids with firstsequences that are complementary to nucleic acids coupled to bindingcomponents, and may further include nucleic acids with second sequencesthat are complementary to nucleic acids coupled to label components. Thefirst sequences can differ from the second sequences such that thedifferent types of components are appropriately directed to a subset ofthe coupling sites. Alternatively, the first and second sequences can bethe same. Optionally, a retaining component may include one or morefirst functional groups that are configured to form covalent bonds withfunctional groups coupled to binding components, and may further includeone or more second functional groups that are configured to formcovalent bonds with functional groups coupled to label components. Thefirst functional group(s) can differ from the second functional group(s)such that the different types of components are appropriately directedto a subset of the coupling sites. For example, the first plurality offunctional groups may participate in a bond-forming reaction that isorthogonal to the bond-forming reaction in which the second plurality offunctional groups participates. Alternatively, the first and secondfunctional groups can be the same. In some configurations, a retainingcomponent may include a mixture of attachment site types, such asnucleic acids and functional groups, where each type of attachment siteis configured to attach a different type of component.

A retaining component may include one or more other components (e.g. abinding component, label component or another retaining component) thatis attached to the retaining component. A retaining component may beattached to another component by a covalent bond (e.g., via a clickreaction), a coordination bond (e.g., a silane linker to a siliconnanoparticle), or a non-covalent bond (e.g., nucleic acidhybridization). A retaining component may be attached to anothercomponent by a chemical reaction that forms a covalent bond between afunctional group on the retaining component and a functional group onthe other component. The reaction may occur by any suitable method,including nucleophilic substitution, electrophilic substitution, andelimination reactions. In some configurations, a covalent bond between aretaining component and another component may be formed by abioorthogonal or click reaction. A retaining component and/or othercomponent may be modified to include a functional group that isconfigured to participate in a bioorthogonal or click reaction.Exemplary functional groups and linkages that may be used to attach aretaining component to one or more other components are set forth infurther detail herein, for example, in the context of making detectableprobes or affinity reagents.

In some configurations, a retaining component may include a nucleic acidstructure, such as a SNAP. In some cases, the nucleic acid structure mayinclude regions of single-stranded nucleic acid that provide targeted,site-specific hybridization sites for the attachment of other components(e.g. a binding component, label component or another retainingcomponent) to the retaining component. In such configurations, othercomponents attached to complementary oligonucleotides can be annealed tosingle stranded sequences on a retaining component to form an attachmentat the targeted sites on the retaining component. In otherconfigurations, oligonucleotides including functional groups may beannealed to a retaining component, thereby permitting subsequentattachment of another component via a chemical reaction (e.g., a clickreaction) between the functional group on the oligonucleotide and afunctional group on the other component.

A detectable probe or affinity reagent may include a retaining componentattached to a plurality of binding components. The plurality of bindingcomponents attached to the retaining component may be chosen to increasethe avidity of the detectable probe or affinity reagent. In someconfigurations, a detectable probe or affinity reagent may contain aplurality of binding components that is homogeneous with regard tospecies (e.g., only antibodies, only aptamers, only nanobodies, onlymini-peptide binders, only DARPins etc.). In other configurations, adetectable probe or affinity reagent may contain a plurality of bindingcomponents that is heterogeneous with regard to species (e.g., a mixtureof antibodies, nanobodies, mini-protein binders, DARPins and/oraptamers). In some configurations, a detectable probe or affinityreagent may contain a plurality of binding components that haveessentially the same binding specificity for a particular bindingpartner, epitope, or target moiety. In other configurations, adetectable probe or affinity reagent may contain a plurality of bindingcomponent that have a mixture of binding specificities for a particularbinding partner, epitope, or target moiety.

The quantity and/or variety of binding components attached to adetectable probe or affinity reagent may be chosen to increase theavidity of the detectable probe or affinity reagent. A detectable probeor affinity reagent may have a total of at least about 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, ormore, whether being different from each other (e.g. a heterogeneousmixture of binding components) or the same (e.g. a homogenous set ofbinding components). Alternatively or additionally, a detectable probeor affinity reagent may have a total of no more than about 100, 95, 90,85, 80, 75, 70, 65, 60, 55, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40,39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22,21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, orless, whether being different from each other or the same.

A detectable probe or affinity reagent may include a heterogeneousmixture of binding components at a chosen ratio, heterogeneity beingbased upon binding component species and/or binding specificity. Forexample, a detectable probe or affinity reagent for a particular epitopemay include a high specificity binding component and a mediumspecificity binding component at a ratio of about 3:1 on a molar basis.A detectable probe or affinity reagent may include a first bindingcomponent and a differing second binding component at a ratio of atleast about 1:1, 1.5:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1,25:1, 50:1, 100:1, 250:1, 500:1, 1000:1, 2500:1, 5000:1, 10000:1,25000:1, 50000:1, 100000:1, 250000:1, 500000:1, 1000000:1, or more.Alternatively or additionally, a detectable probe or affinity reagentmay include a first binding component and a differing second bindingcomponent at a ratio of no more than about 1000000:1, 500000:1,250000:1, 100000:1, 50000:1, 25000:1, 10000:1, 5000:1, 2500:1, 1000:1,500:1, 250:1, 100:1, 50:1, 25:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1,3:1, 2:1, 1.5:1, or less.

An affinity reagent, probe or binding component of the presentdisclosure may have a characterized binding probability for a bindingpartner, epitope, or target moiety. For example, an affinity reagent,probe or binding component may be known to bind to a certain polypeptideepitope and may be known to show a high probability of binding (e.g.,fails to show evidence of binding 1 out of every 100 observations). Inanother example, an affinity reagent, probe or binding component may becharacterized as binding to a certain epitope with a low, but non-zero,binding probability (e.g., 0.00001% chance of binding for a givenobservation). A probabilistic characterization may include two aspectsregarding binding probability: 1) the structure-dependent likelihood ofbinding to a binding partner, epitope, or target moiety; and 2) theenvironmentally-dependent likelihood of binding to a binding partner,epitope, or target moiety.

The binding probability of an affinity reagent, probe or bindingcomponent for a binding partner, epitope, or target moiety may beextended beyond polypeptides to include non-polypeptide or heterogeneoussystems (e.g., soil, crude cell lysates, bodily fluids). For example, anaffinity reagent, probe or binding component may have a characterizedbinding probability for a component of a composite (e.g., metalnanoparticles embedded in a polymer matrix) or may preferentially bindto structural subunits within a polysaccharide (e.g., glycosylations,hemicelluloses, celluloses, lignins, pectins, etc.). The skilled personwill recognize from the present disclosure that an affinity reagent,probe or binding component intended for a non-polypeptide orheterogeneous system may show the analogous property of possessing anon-zero probability of binding to a non-polypeptide binding partner,epitope, or target moiety for which the affinity reagent, probe orbinding component possesses a low binding affinity.

The binding affinity or binding promiscuity of an affinity reagent,probe or binding component may pertain to the effect that polypeptideprimary, secondary, tertiary, or quaternary structure (i.e. amino acidsequence) has on binding. For example, an affinity reagent, probe orbinding component may be characterized as binding with increased ordecreased preference for particular polypeptide epitope sequences (e.g.,amino acid trimers, tetramers, pentamers, etc.). The structure-dependentlikelihood of an affinity reagent, probe or binding component binding toan epitope may also be affected by sequence context (e.g., amino acidsthat flank the amino terminus and/or carbonyl terminus of a peptideepitope; amino acid residues that are proximal to a peptide epitope inthe secondary or tertiary structures of a polypeptide, the presence orabsence of post-translational modifications in or around a peptideepitope, etc.). An affinity reagent, probe or binding component of thepresent disclosure may have substantial affinity or promiscuity for afamily of amino acid epitopes (e.g., AXA, where A represents alanine andX represents any of the 20 naturally-occurring amino acids). Thestructure-dependent likelihood of binding may be calculated for eachaffinity reagent, probe or binding component used for polypeptidecharacterization, such as by an empirical binding model or a database ofbinding probabilities. An affinity reagent, probe or binding componentmay have a sequence-specific likelihood of binding to a binding partner,epitope, or target moiety of at least about 0.000001%, 0.00001%,0.0001%, 0.001%, 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, 99.999%, 99.9999%,99.99999%, 99.999999% or more. Alternatively or additionally, anaffinity reagent, probe or binding component may have asequence-specific likelihood of binding to a binding partner, epitope,or target moiety of no more than about 99.999999%, 99.99999%, 99.9999%,99.999%, 99.99%, 99.9%, 99.5%, 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%,75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%,5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, 0.001%, 0.0001%, 0.00001%,0.000001%, or less.

The environmentally-dependent likelihood of binding an affinity reagent,probe or binding component to an epitope in a polypeptide may pertain tothe effect of variables other than the structure of the epitope and/orthe polypeptide on binding. For example, binding of an affinity reagent,probe or binding component to a particular epitope may vary based uponsolvent chemical composition (e.g., solvent identity, solvent polarity,ionic strength of the solvent, buffer concentration, pH, presence ofsurfactants or denaturants, etc.). Other non-polypeptide variables mayinclude the time duration of binding; concentration of affinity reagent,probe or binding component; concentration of binding partner, epitope ortarget moiety; and the presence of externally-applied fields, such asheat, electrical fields, magnetic fields, and fluid velocity fields. Anaffinity reagent, probe or binding component may have anenvironmentally-dependent likelihood of binding to a binding partner,epitope, or target moiety of at least about 0.000001%, 0.00001%,0.0001%, 0.001%, 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, 99.999%, 99.9999%,99.99999%, 99.999999% or more. Alternatively or additionally, anaffinity reagent, probe or binding component may have anenvironmentally-dependent likelihood of binding to a binding partner,epitope, or target moiety of no more than about 99.999999%, 99.99999%,99.9999%, 99.999%, 99.99%, 99.9%, 99.5%, 99%, 98%, 97%, 96%, 95%, 90%,85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%,15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, 0.001%, 0.0001%,0.00001%, 0.000001%, or less.

In some configurations, the structure-dependent binding likelihood andthe environmentally-dependent binding likelihood may be combined todetermine an overall likelihood or probability of an affinity reagent,probe or binding component binding to a binding partner, epitope, ortarget moiety. Overall likelihoods or probabilities may be compiled forsome or all known binding partners, epitopes, or target moieties tocreate a probabilistic binding profile for an affinity reagent, probe orbinding component. In some configurations, an affinity agent, probe orbinding component may be characterized as binding to a set of N bindingpartners, epitopes, or target moieties with an overall bindingprobability of at least about 20%, and a set of M binding partners,epitopes, or target moieties with an overall binding probability of nomore than 0.1%, where N≥1, M≥1, and M≥10 N. An affinity reagent, probeor binding component may have an overall likelihood or probability ofbinding to a binding partner, epitope, or target moiety of at leastabout 0.000001%, 0.00001%, 0.0001%, 0.001%, 0.01%, 0.1%, 0.5%, 1%, 2%,3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%,99.999%, 99.9999%, 99.99999%, 99.999999% or more. Alternatively oradditionally, an affinity reagent, probe or binding component may havean overall likelihood or probability of binding to a binding partner,epitope, or target moiety of no more than about 99.999999%, 99.99999%,99.9999%, 99.999%, 99.99%, 99.9%, 99.5%, 99%, 98%, 97%, 96%, 95%, 90%,85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%,15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, 0.001%, 0.0001%,0.00001%, 0.000001%, or less.

A retaining component may include one or more label components that areassociated with the retaining component. In some configurations, the oneor more label components may be attached to the retaining component ofthe detectable probe or affinity reagent. In some configurations, theone or more label components may be non-covalently associated with thedetectable probe (e.g., nucleic acid intercalation dyes) or affinityreagent. In some configurations, a retaining component may surround orenclose a label component (e.g., a polymer coating on a FluoSphere™ orquantum dot). A label component may be attached to a retaining componentby a covalent bond (e.g., via a click reaction), a coordination bond(e.g., a silane linker to a silicon nanoparticle), or a non-covalentbond (e.g., nucleic acid hybridization). A label component may beattached to a retaining component by a chemical reaction that forms acovalent bond between a functional group on the retaining component anda functional group on the label components. The reaction may occur byany suitable method, including those set forth herein or known in theart.

A retaining component may include a particle (e.g. microparticle ornanoparticle) that provides one or more attachment sites for othercomponents (e.g. a binding component, label component or anotherretaining component). In some configurations, a particle may include asurface that is functionalized, can be functionalized, or is otherwisemodifiable to provide attachment sites for other components. In someconfigurations, a particle may provide a template for a shell or coating(e.g., a polymer or hydrogel coating) that contains or can be modifiedto contain attachment sites for components. In some configurations, aretaining component may effectively function as a label component (e.g.,a FluoSphere™ or quantum dot). A particle may include a surrounding orconcentric shell, layer, or coating that provides attachment sites ormodifiable sites for the attachment of components. The shell, coating,or layer may include a polymer or hydrogel that has been covalently ornon-covalently joined to the particle surface. The shell, coating, orlayer may include a plurality of functional groups that can form acovalent bond or can be modified to form a covalent bond with anothermolecule. The shell, coating, or layer may be modified with additionalgroups that provide attachment sites or otherwise modify the surface(e.g., providing steric hindrance by PEGylation of the surface).

Any of a variety of detectable moieties may be used as label componentsfor an affinity reagent or detectable probe described herein. Forexample, in some cases, the labels may be electrochemical labels and maybe detected by electrochemical detection. These may be charged moieties,for example large nucleic acids, ploylysine, and other highly chargedchemical structures, and detection regions may be ChemFET type sensors.Alternatively or additionally, in some cases, the detectable moietiesmay be optically detectable moieties, i.e., detectable based uponobservation of differential light energy that comes from the label.

A detectable probe may include one or more retaining components attachedto one or more label components. The one or more label componentsattached to the retaining component may be chosen to increase theobservability of the detectable probe. In some configurations, adetectable probe may contain a plurality of label components that arehomogeneous with regard to species of label (e.g., a single type offluorophore, homogeneous nucleic acid barcodes, etc.). In otherconfigurations, a detectable probe may contain a heterogeneous pluralityof label component species (e.g., a mixture of fluorophores withdiffering emissions wavelengths, a mixture of fluorophores and nucleicacid barcodes). Optionally, a detectable probe may contain a pluralityof label components that produce overlapping signals or signals that areindistinguishable when detected in a method or apparatus set forthherein. For example, a plurality of fluorophores present in a detectableprobe may emit fluorescence at a common wavelength whether thefluorophores are excited at the same wavelength as each other ordifferent wavelengths form each other. Alternatively, a detectable probemay contain a plurality of label components that produce differentsignals from each other, for example, signals that are distinguishableor distinguished when detected in a method or apparatus set forthherein.

A detectable label or label component may produce a detectable signalthat permits identification of an interaction between an affinityreagent (e.g. detectable probe) and a binding partner, epitope, ortarget moiety. A detectable label or label component may be configuredto provide spatial information, such as providing a detectable signal ata spatially-resolved location. A detectable label or label component maybe configured to provide temporal information, such as providing anevanescent or decaying signal, optionally at a spatially-resolvedlocation. A detectable label or label component may emit a detectablesignal in the presence of an excitation source (e.g., radiation, heat, achemical substrate). A detectable label or label component may emit adetectable signal in the absence of an excitation source (e.g., aradiolabel or chemiluminescent label). A detectable label or labelcomponent may contain an encoded signal, such as a nucleic acid orpolypeptide barcode.

In some configurations, a label component may include an attachedenzyme, protein, or a sequence of enzymes that create a detectablechemical signal. Exemplary enzymes may include horseradish peroxidase(HRP) or alkaline phosphatase. An enzyme or protein may be chosen thatconverts a substrate molecule into a detectable molecule (e.g., afluorescent compound) or binds a substrate molecule that in turnproduces a fluorescent or luminescent effect in the enzyme or protein.For example, HRP may convert a substrate molecule such as ABTS, OPD,AmplexRed, DAB, AEC, TMB, homovanillic acid, or luminol into afluorescent or luminescent molecule. In some configurations, adetectable probe or affinity reagent may interact with a bindingpartner, epitope, or target moiety in the presence of a substratemolecule to produce a deposited fluorescent or luminescent molecule thatprovides a spatial signal of the location of the detectable probe oraffinity reagent binding interaction. In an alternative configuration, adetectable probe or affinity reagent binding interaction may be detectedby a reactive pathway or reaction sequence that produces a detectablesignal through a succession of reactions of a substrate molecule. Forexample, an enzyme such as HRP or alkaline phosphatase may becolocalized with a binding partner, epitope, or target moiety. Adetectable probe or affinity reagent may include one or more enzymesthat convert a substrate molecule from a substrate that cannot beprocessed or altered by the colocalized enzyme to a product that is asubstrate for the colocalized enzyme. A detectable probe or affinityreagent may include a plurality of differing enzymes or an enzymaticcomplex (e.g., a polyketide synthase) that converts a substrate into adetectable product.

A detectable label or label component may be detected by a signaldetection source as would be appropriate for the chosen label species.Optical labels and optical detectors are particularly useful. Examplesof optical detection apparatus and components thereof that can be usedherein include those commercialized for nucleic acid sequencing such asthose provided by Illumina™, Inc. (e.g. HiSeq™, MiSeq™, NextSeq™, orNovaSeq™ systems), Life Technologies™ (e.g. ABI PRISM™, or SOLID™systems), Pacific Biosciences (e.g. systems using SMRT™ Technology suchas the Sequel™ or RS II™ systems), or Qiagen (e.g. Genereader™ system)or those described in US Pat. App. Pub. No. 2010/0111768 A1 or U.S. Pat.Nos. 7,329,860; 8,951,781 or 9,193,996, each of which is incorporatedherein by reference. Other useful detectors are described in U.S. Pat.Nos. 5,888,737; 6,175,002; 5,695,934; 6,140,489; or 5,863,722; or USPat. Pub. Nos. 2007/007991 A1, 2009/0247414 A1, or 2010/0111768; orWO2007/123744, each of which is incorporated herein by reference in itsentirety.

Other detection techniques that can be used in a method set forth hereininclude, for example, mass spectrometry which can be used to perceivemass; surface plasmon resonance which can be used to perceive binding toa surface; absorbance which can be used to perceive the wavelength ofthe energy a label absorbs; calorimetry which can be used to perceivechanges in temperature due to presence of a label; electricalconductance or impedance which can be used to perceive electricalproperties of a label, or other known analytic techniques. For example,charged labels can be detected using electronic detectors such aschemFET detectors used for detection of protons or pyrophosphate (see,for example, US Pat. App. Pub. Nos. 2009/0026082 A1; 2009/0127589 A1;2010/0137143 A1; or 2010/0282617 A1, each of which is incorporatedherein by reference in its entirety, or detectors commercialized in theIon Torrent™ systems commercially available from ThermoFisher, Waltham,Mass.) A FET detector can be used such as one or more of those describedin US Pat. App. Pub. Nos. 2017/0240962 A1, 2018/0051316 A1, 2018/0112265A1, 2018/0155773 A1 or 2018/0305727 A1; or U.S. Pat. Nos. 9,164,053,9,829,456, 10,036,064, or 10,125,391, each of which is incorporatedherein by reference.

A label component may include a moiety or molecule that is luminescent(e.g. a luminophore or fluorophore). Luminophores, which emit light ofone wavelength in response to being excited by a light of anotherwavelength, are particularly useful as optically detectable labels giventheir ability to provide readily detectable light signals, as well astheir ability to be tailored to a variety of different excitation andemission light spectra, providing great flexibility in their deploymentand use.

Any of a variety of luminophores may be used herein. In some cases, theluminophore may be a small molecule. In some cases, the luminophore maybe a protein. Luminophores may include labels that emit in theultraviolet spectrum, visible spectrum, or infrared spectrum. In somecases, the luminophore may be selected from the group consisting ofFITC, Alexa Fluor® 350, Alexa Fluor® 405, Alexa Fluor® 488, Alexa Fluor®532, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor®594, Alexa Fluor® 647, Alexa Fluor® 680, Alexa Fluor® 750, Pacific Blue,Coumarin, BODIPY FL, Pacific Green, Oregon Green, Cy3, Cy5, PacificOrange, TRITC, Texas Red, R-Phycoerythrin, Allophcocyanin (APC). In somecases, the label may be an Atto dye, for example Atto 390, Atto 425,Atto 430, Atto 465, Atto 488, Atto 490, Atto 495, Atto 514, Atto 520,Atto 532, Atto 540, Atto 550, Atto 565, Atto 580, Atto 590, Atto 594,Atto 610, Atto 611, Atto 612, Atto 620, Atto 633, Atto 635, Atto 647,Atto 655, Atto 680, Atto 700, Atto 725, Atto 740, Atto MB2, Atto Oxa12,Atto Rho101, Atto Rho12, Atto Rho13, Atto Rho14, Atto Rho3B, Atto Rho6G,or Atto Thio12. In some cases, the luminophore may be a fluorescentprotein, for example a fluorescent protein selected from the groupconsisting of green fluorescent protein (GFP), cyan fluorescent protein(CFP), red fluorescent protein (RFP), blue fluorescent protein (BFP),orange fluorescent protein (OFP), and yellow fluorescent protein (YFP).A wide range of effective luminophores are commercially available, forexample, from the Molecular Probes division of ThermoFisher Scientificand/or generally described in the Molecular Probes Handbook (11^(th)Edition) which is hereby incorporated by reference. Label components mayalso include intercalation dyes, such as ethidium bromide, propidiumbromide, crystal violet, 4′,6-diamidino-2-phenylindole (DAPI),7-aminoactinomycin D (7-AAD), Hoescht 33258, Hoescht 33342, Hoescht34580, YOYO-1, DiYO-1, TOTO-1, DiTO-1, or combinations thereof.

Specific fluorescent labels or other luminophores may be chosendepending upon the desired use, and in some cases, selected based ontheir absorption/emission spectra, for example, to optimize multiplexeddetection. Likewise, in some cases, luminophores may include pairs offluorescent dyes that interact to provide advantageous fluorescentproperties. For example paired dyes can function as Forster resonantenergy transfer (FRET) pairs, where excitation of one member of the pair(i.e. “donor”) results in an energy transfer that excites or istransferred to the other member (i.e. “acceptor”). The acceptor thenemits luminescence at a wavelength that is shifted from the emissionthat would have occurred from the donor alone.

In some cases, optically detectable labels may include other types ofdetectable moieties. For example, in some cases, luminescent particles(e.g. microparticles or nanoparticles), such as semiconductornanoparticles, “quantum dots”, or FluoSphere™ particles, may be includedas label components.

A luminophore may be characterized by a characteristic excitation orabsorbance wavelength. An excitation source may include a light sourcethat is tuned to a characteristic excitation or absorbance wavelength ofa luminophore. A luminophore may absorb light over a range ofwavelengths, with maximum absorbance of light occurring at a peakwavelength. A luminophore can be excited at or near peak excitation. Inparticular configurations of methods set forth herein a luminophore canbe excited by radiation in the ultraviolet (UV), visible (VIS) orinfrared (IR) region of the spectrum. Excitation in the VIS region mayoccur at one or more of the red, orange, yellow, green, blue or violetregions of the spectrum. A luminophore may be characterized by acharacteristic emission wavelength. A luminophore may emit light over arange of wavelengths, with maximum emission of light occurring at a peakwavelength. A luminophore can be detected at or near peak emission. Inparticular configurations of methods set forth herein emission from aluminophore can be detected in the ultraviolet (UV), visible (VIS) orinfrared (IR) region of the spectrum. Detection in the VIS region mayoccur at one or more of the red, orange, yellow, green, blue or violetregions of the spectrum.

The presence of multiple label components in a detectable probe mayincrease the observability of the detectable probe, for example, by 1)increasing the likelihood of detection; 2) providing redundancy in caseof label loss (e.g., photobleaching, chemical damage, cleavage oflabels, etc.); and/or 3) increasing the signal strength generated by adetectable probe. The quantity of label components associated with adetectable probe may be decided by various factors, including probesize, desired label spacing, signal intensity, measurement length,measurement environments, and label size.

A detectable probe may have a chosen number of associated labelcomponents. A detectable probe may have at least about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, ormore, whether being different from each other (e.g. a heterogeneousmixture of labels that produce signals that are distinguishable fromeach other in a method or apparatus set forth herein) or the same (e.g.a mixture of labels that produce signals that are indistinguishable in amethod or apparatus set forth herein). Alternatively or additionally, adetectable probe may have no more than about 100, 95, 90, 85, 80, 75,70, 65, 60, 55, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37,36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19,18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or less,whether being different from each other (e.g. a heterogeneous mixture oflabels that produce signals that are distinguishable from each other ina method or apparatus set forth herein) or the same (e.g. a mixture oflabels that produce signals that are indistinguishable in a method orapparatus set forth herein).

The chosen variety or quantity of label components on a detectable probemay provide a signal that exceeds a background signal. For example,probes that are detected by fluorescence may produce a fluorescentsignal that exceeds the existing background from sources such asautofluorescence, signal cross-talk, and impinging external sources. Adetectable probe may include a variety or quantity of label componentsthat is configured to produce a detectable signal intensity that exceedsa signal background intensity (e.g., maximum, minimum, or average) by atleast 2×, 5×, 10×, 25×, 50×, 100×, or more. Additionally oralternatively, a detectable probe may include a quantity of labelcomponents that is configured to produce a detectable signal intensitythat exceeds a signal background intensity (e.g., maximum, minimum, oraverage) by no more than about 100×, 50×, 25×, 10×, 5×, 2×, or less.

A detectable probe may include more than one type and/or species oflabel component. For example, a detectable probe may include at leastone luminophore and at least one nucleic acid barcode sequence. Inanother example, a detectable probe may include two or more differentluminophores (e.g., Alexa-Fluor® 488 and Alexa-Fluor® 647). Thedifferent luminophores can differ with respect to one or more signalproperties such as their excitation spectra, emission spectra,luminescence lifetime, or luminescence polarity. In some configurationsthe different luminophores can be similar with regard to one or moresignal properties. For example, two luminophores can be excited at thesame wavelength, but emit at different wavelengths. As such, a singleexcitation source can be used to excite two different luminophores thatare nonetheless distinguished based on differences in their emissionproperties. A detectable probe may include more than one type and/orspecies of label component for various purposes, including creatingunique signal fingerprints, enabling multi-label detection methods(e.g., FRET or luminescence quenching), and/or generating signalredundancy to reduce false positive or false negative detections.

A detectable probe may include a heterogeneous mixture of labelcomponents, based upon label type and/or species. A detectable probe mayhave at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100, or more label components, whether beingthe same as each other or different from each other. Alternatively oradditionally, a detectable probe may have no more than about 100, 95,90, 85, 80, 75, 70, 65, 60, 55, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41,40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23,22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3,or fewer label components, whether being the same as each other ordifferent from each other.

A detectable probe may include a heterogeneous mixture of labelcomponents at a chosen ratio, based upon detectable label species. Forexample, a detectable probe for a particular epitope may includeAlexa-Fluor® 488 and Alexa-Fluor®-647 dyes at a ratio of about 3:1 on amolar basis, respectively. A detectable probe may include a first labelcomponent and a differing second label component at a ratio of at leastabout 1:1, 1.5:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 25:1,50:1, 100:1, 250:1, 500:1, 1000:1, 2500:1, 5000:1, 10000:1, 25000:1,50000:1, 100000:1, 250000:1, 500000:1, 1000000:1, or more. Alternativelyor additionally, a detectable probe may include a first label componentand a differing second label component at a ratio of no more than about1000000:1, 500000:1, 250000:1, 100000:1, 50000:1, 25000:1, 10000:1,5000:1, 2500:1, 1000:1, 500:1, 250:1, 100:1, 50:1, 25:1, 10:1, 9:1, 8:1,7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1.5:1, or less.

FIG. 49A provides a schematic representation of a detectable probe. Eachdetectable probe consists of two parts: the labeled affinity probe (200)and template (201). As shown, the labeled affinity probe 200 includes abinding component 200 a, an annealing region 200 b which provides aretaining component, and an enzymatically extended label component 200c. The template 201 includes 201 a, a complimentary sequence toannealing region 200 b, and 201 b, the template for enzymatic extensionof 200 c. FIG. 49B shows a step by step demonstration of enzymaticextension off of an aptamer probe to form the label component.

In some cases, a label component may include or be attached to aretaining component (e.g. a scaffold as set forth in U.S. ProvisionalApplication No. 63/112,607). Multiple label components may be providedrandomly spaced along the retaining component, or in some cases, may bepositioned at regular or semi-regular intervals along the length of theretaining component. In other cases, a single label component may beattached to a retaining component, e.g., at the 5′ terminus of a nucleicacid, see FIG. 50A. In yet other cases, a nucleic acid of a retainingcomponent can be designed to include an annealing region, and may have asingle label component (e.g. a fluorophore) at the end of the annealingregion. A second strand of nucleic acid with an additional singlefluorophore can anneal to this region, see FIG. 50B.

It will be understood that an affinity reagent of the present disclosureneed not include any label components. Accordingly, an affinity reagentcan be configured to omit or be devoid of one or more label componentsor species of label components set forth herein. Moreover, exemplaryaffinity reagents or detectable probes set forth herein as having one ormore label components may be reconfigured to omit one or more of theexemplified label components. It will also be understood that anaffinity reagent or detectable probe of the present disclosure can beconfigured to omit or be devoid of one or more binding components orspecies of binding components set forth herein.

A retaining component may form a central structural element of adetectable probe or affinity reagent. In some configurations, aretaining component may be structured to provide constraint or controlof the physical positioning of other components, such as bindingcomponents, label components or other retaining components. For example,a detectable probe or affinity reagent may include binding componentsthat are attached at sufficiently spaced positions on a retainingcomponent to prevent contact or cross-reactivity between the bindingcomponents. In another example, a detectable probe may includeluminophores that are attached at sufficiently spaced positions on aretaining component to reduce or prevent quenching between adjacentluminophores. In a third example, a detectable probe may includeluminophores that are attached at positions on a retaining componentthat are spaced and oriented to facilitate FRET. A retaining componentmay be engineered or rationally designed to control the location and/orpositioning of components attached thereto. Factors that may influencethe design of a retaining component include: 1) nature of a likelyinteraction between adjacent components; 2) likelihood of interactionbetween adjacent components; 3) critical scales (e.g., distance, volume,time) for likely interactions between adjacent components; 4) physicalproperties of the retaining component (e.g., shape, conformation, size,rigidity, etc.); 5) physical properties of the attached probe components(shape, conformation, size, hydrodynamic radius, etc.); 6) nature andproperties of optional linkers that bind components to the retainingcomponent; and 7) nature of a likely interaction between a detectableprobe or affinity reagent and a binding partner.

In some configurations, a detectable probe or affinity reagent mayinclude a retaining component including one or more nucleic acids. Forexample, the one or more nucleic acids that form the retaining componentmay take the form of a nucleic acid origami structure, nucleic acidnanoball structure or other structured nucleic acid particle. A nucleicacid retaining component can contain a single nucleic acid strand or aplurality of nucleic acid strands, such as a plurality ofoligonucleotide strands. A retaining component including a plurality ofnucleic acid strands may be formed by hybridization between theplurality of strands to form nucleic acid structures with increasedstructural complexity beyond the natural helical structure. A nucleicacid may include nucleic acids such as DNA, RNA, PNA, or combinationsthereof. Nucleic acids may include non-natural nucleotides, such asluminescently modified nucleotides. Nucleic acids may includenon-nucleotide residues within their structures such as photocleavablelinkers (e.g., nitrobenzyl, carbonyl, or benzyl-based photocleavablelinker). A retaining component including a nucleic acid may bestructured to break up or destabilize under certain conditions. Aretaining component may be broken up or destabilized to, for example,facilitate removal of a detectable probe or affinity reagent from abinding partner. A retaining component including a nucleic acid mayinclude a plurality of restriction sites that can be cleaved by arestriction enzyme, thereby facilitating break up or destabilization ofthe nucleic acid retaining component. A retaining component including anucleic acid may include a plurality of photocleavable, chemicallycleavable or otherwise reactive bonds, thereby facilitating break up ordestabilization of the retaining component.

A retaining component can include a nucleic acid nanoball. The nanoballcan contain a single strand of nucleic acid that folds in on itself toform a compact structure. Optionally, the nanoball can be crosslinked toconstrain the nanoball to a relatively compact structure. Exemplarycrosslinks include chemical crosslinks such as psoralen oroligonucleotides that hybridize to different regions of the singlestrand. Nucleic acid nanoballs can be created by rolling circleamplification of a circular template to yield a concatemericamplification product in which each unit of sequence in the concatemerhas a sequence that complements the circular template. Exemplarysequence elements that can be incorporated into a nucleic acid nanoballinclude tag sequences that provide information about the nanoball suchas its source or history of use, sequences that complementoligonucleotides used as intrastrand crosslinkers, sequences thatcomplement oligonucleotides that are attached to functional groups orcomponents such as binding components or label components, or the like.Exemplary nucleic acid nanoballs and methods for their manufacture anduse are set forth in U.S. Pat. No. 8,445,194, which is incorporatedherein by reference.

A nucleic acid nanoball may be formed by a method such as rolling circleamplification (RCA) or ligation of repeated concatemer units. A nucleicacid nanoball may include a particular number of repeated concatemerunits. A nucleic acid nanoball may include at least about 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150,160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800,900, 1000 or more concatemer units. Alternatively or additionally, anucleic acid nanoball may include no more than about 1000, 900, 800,700, 600, 500, 450, 400, 350, 300, 250, 200, 190, 180, 170, 160, 150,140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40,35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, or fewer concatemer units. A concatemer unit in a nucleic acidnanoball may have a nucleotide sequence length of at least about 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120,130, 140, 150, 160, 170, 180, 190, 200, or more nucleotides.Alternatively or additionally, a concatemer unit in a nucleic acidnanoball may have a nucleotide sequence length of no more than about200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80,75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, or fewer nucleotides.

A retaining component can include a nucleic acid origami. Accordingly, aretaining component can include one or more nucleic acids havingtertiary or quaternary structures such as spheres, cages, tubules,boxes, tiles, blocks, trees, pyramids, wheels, combinations thereof, andany other possible structure. Examples of such structures formed withDNA origami are set forth in Zhao et al. Nano Lett. 11, 2997-3002(2011), which is incorporated herein by reference. In someconfigurations, a nucleic acid origami may include a scaffold and aplurality of staples, where the scaffold is a single, continuous strandof nucleic acid, and the staples are oligonucleotides that areconfigured to hybridize, in whole or in part, with the scaffold nucleicacid. Examples of nucleic acid origami structures formed using acontinuous scaffold strand and several staple strands are set forth inRothemund Nature 440:297-302 (2006) or U.S. Pat. No. 8,501,923 or U.S.Pat. No. 9,340,416, each of which is incorporated herein by reference. Aretaining component including one or more nucleic acids (e.g. as foundin origami or nanoball structures) may include regions ofsingle-stranded nucleic acid, regions of double-stranded nucleic acid,or combinations thereof.

In some embodiments, a nucleic acid origami includes a scaffold having aclosed nucleic acid strand, and a plurality of oligonucleotideshybridized to the scaffold. A nucleic acid scaffold may include acontinuous strand of nucleic acids that, absent complementaryoligonucleotides, is a circular or joined strand (i.e., no 5′ or 3′termini). In some configurations, a nucleic acid scaffold is derivedfrom a natural source, such as a viral genome or a bacterial plasmid. Inother configurations, a nucleic acid scaffold may be engineered,rationally designed, or synthetic. A scaffold may include one or moremodified nucleotides. Modified nucleotides may provide functional groupsor attachment sites for attaching additional components (e.g. bindingcomponent, label component or another retaining component) before,during, or after assembly of a detectable probe or affinity reagent. Amodified nucleotide may include a linking group or a functional group(e.g., a functional group configured to perform a click reaction). Insome configurations, a nucleic acid scaffold may include a single strandof an M13 viral genome. The size of a nucleic acid scaffold may varydepending upon the desired size of the retaining component. A nucleicacid scaffold may include at least about 1000, 2000, 3000, 4000, 5000,6000, 7000, 8000, 9000, 10000, or more nucleotides. Alternatively oradditionally, a nucleic acid scaffold may include at most about 10000,9000, 8000, 7000, 6000, 5000, 4000, 3000, 2000, 1000 or fewernucleotides.

A retaining component, such as a nucleic acid origami, may include aplurality of oligonucleotides (e.g. staples). Staples may includeoligonucleotides that are configured to hybridize with a nucleic acidscaffold, other staples, or a combination thereof. A staple may includeone or more modified nucleotides. Staples may be modified to includeadditional chemical entities, such as binding components, labelcomponents, chemically-reactive groups (e.g. functional groups orhandles), or other groups (e.g., polyethylene glycol (PEG) moieties).Staples may include linear or circular nucleic acids. Staples mayinclude regions of single-stranded nucleic acids, double-strandednucleic acids, or combinations thereof. A staple may be configured tobind with other nucleic acids by complementary base pair hybridizationor ligation. A staple may be configured to act as a primer for acomplementary nucleic acid strand and the priming staple may be extendedby an enzyme (e.g., a polymerase such as a template directed polymeraseor non-template directed polymerase such as terminal transferase) toform lengthened regions of double-stranded nucleic acid.

A staple may be any length depending upon the design of the retainingcomponent. Staples may be designed by a software package, such asCADNANO, ATHENA, or DAEDALUS. A staple may have a length of at leastabout 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600,650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500,1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, 5000, ormore nucleotides. Alternatively or additionally, a staple may have alength of no more than about 5000, 4500, 4000, 3500, 3000, 2500, 2000,1900, 1800, 1700, 1600, 1500, 1400, 1300, 1200, 1100, 1000, 950, 900,850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200,150, 100, 50, 25, 10, or fewer nucleotides.

A staple may include one or more modified nucleotides. Modifiednucleotides may provide attached sites for attaching additionalcomponents, such as binding components or label components. A modifiednucleotide may be utilized as an attachment site for an additionalcomponent before, during, or after assembly of a detectable probe oraffinity reagent. A staple may include at least about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 20, 30, 40, 50, 75, 100 or more modified nucleotides.Alternatively or additionally, a staple may include no more than about100, 75, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 modifiednucleotides.

A staple may be designed or modified to achieve desired stability of adetectable probe, affinity reagent or nucleic acid origami. Stabilitymay be affected by the dissociation of individual oligonucleotides fromthe assembled origami. Loss of oligonucleotides could have variousdestabilizing effects, including loss of functionality for a detectableprobe or affinity reagent (e.g., loss of component attachment sites,loss of binding components, loss of label components, etc.) ordestabilization of secondary or tertiary structures, thereby promotingfurther destabilization of other oligonucleotides. A scaffold or staplein a nucleic acid retaining component may include one or more modifiednucleotides that are configured to form covalent or non-covalent bondsthat promote the stability of the nucleic acid retaining component. Forexample, an oligonucleotide may include one or more modified nucleotidesthat form covalent bonds or cross-links with modified nucleotides inother oligonucleotides or in the scaffold strand. Alternatively oradditionally, an oligonucleotide may be designed to have a minimumhybridization length or minimum melting temperature to decrease thelikelihood of dissociation.

An oligonucleotide in a detectable probe or affinity reagent mayhybridize with another oligonucleotide or a scaffold strand forming aparticular number of base pairs. An oligonucleotide may formahybridization region of at least about 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, or more consecutive or total base pairs. Alternatively oradditionally, an oligonucleotide may form a hybridization region of nomore than about 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37,36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19,18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or fewer consecutiveor total base pairs.

An oligonucleotide in a detectable probe, affinity reagent or nucleicacid origami may have a characterized melting temperature. The meltingtemperature may refer to the temperature at which nucleotide base-pairbinding interactions become interrupted, thereby causing dissociation ofthe oligonucleotide. An oligonucleotide in a nucleic acid retainingcomponent may have a characterized melting temperature of at least about50° C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C., 58° C.,59° C., 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 66° C., 67° C.,68° C., 69° C., 70° C., 71° C., 72° C., 73° C., 74° C., 75° C., 76° C.,77° C., 78° C., 79° C., 80° C., 81° C., 82° C., 83° C., 84° C., 85° C.,86° C., 87° C., 88° C., 89° C., 90° C., or higher. Alternatively oradditionally, an oligonucleotide in a nucleic acid retaining componentmay have a characterized melting temperature of no more than about 90°C., 89° C., 88° C., 87° C., 86° C., 85° C., 84° C., 83° C., 82° C., 81°C., 80° C., 79° C., 78° C., 77° C., 76° C., 75° C., 74° C., 73° C., 72°C., 71° C., 70° C., 69° C., 68° C., 67° C., 66° C., 65° C., 64° C., 63°C., 62° C., 61° C., 60° C., 59° C., 58° C., 57° C., 56° C., 55° C., 54°C., 53° C., 52° C., 51° C., 50° C., or lower.

A detectable probe or affinity reagent may include a retaining componentthat is configured to position a plurality of binding components atspecific locations on the retaining component. The relative positioningmay be determined in part by the potential for positive or negativeinteractions between adjacent binding components. For example, someadjacent binding components (e.g., aptamers, peptamers) may be prone tomisfolding or conformational changes when brought into close proximitywith particular species of binding components. Such binding componentsmay benefit from sufficient separation to minimize the likelihood ofsuch negative interactions. In another example, some binding componentsmay experience an increase in avidity when multiple binding componentsare brought within a close proximity. In some configurations, thepositioning of binding components at specific locations on a retainingcomponent may be determined by optimizing a balance between positiveavidity effects and negative inter-affinity reagent interactions.

Positioning of binding components on a detectable probe or affinityreagent may be determined, in whole or in part, by the structuralproperties of a retaining component. In some configurations, retainingcomponents may include an inherently rigid, inelastic, or non-deformablematerial (e.g., carbon or metal nanoparticles) that is not prone todeformation when used in particular compositions or methods, forexample, in solution or on a solid support. In other configurations,retaining components may include a flexible or deformable material(e.g., polymers, nucleic acids, etc.) that is prone to some degree ofdeformation, such as stretching, compression, or bending (e.g.,torsional or lateral bending). The natural deformation of a retainingcomponent may produce conformational changes that increase or decreasethe relative proximity of adjacent binding components attached to theretaining component.

Binding components may be positioned on a retaining component withsufficient separation relative to other binding components such thataffinity reagents do not have overlapping effective occupied volumes.FIG. 3A depicts a schematic of a first binding component 310 and asecond binding component 320 that are attached to a retaining component300. The first binding component 310 has an effective occupied volume315 and the second binding component 320 has an effective occupiedvolume 325. The first binding component 310 and the second bindingcomponent 320 are attached to the retaining component 300 at aseparation distance Δs that is the minimum distance necessary to ensurethat the effective occupied volumes 315 and 325 do not overlap.

In some configurations, binding components may be positioned on aretaining component with sufficient separation relative to other bindingcomponents such that binding components have a separation gap. FIG. 3Bdepicts a schematic of a first binding component 310 and a secondbinding component 320 that are attached to a retaining component 300with a separation gap Δs. The first binding component 310 has aneffective occupied volume 315 and the second binding component 320 hasan effective occupied volume 325. The first binding component 310 andthe second binding component 320 are attached to the retaining component300 at a separation distance Δs that is the minimum distance necessaryto ensure that the effective occupied volumes 315 and 325 have aseparation gap of length Δg.

In some configurations, binding components may be positioned on aretaining component with a separation relative to other bindingcomponents such that binding components have overlapping effectiveoccupied volumes. FIG. 3C depicts a schematic of a first bindingcomponent 310 and a second binding component 320 that are attached to aretaining component 300. The first binding component 310 has aneffective occupied volume 315 and the second binding component 320 hasan effective occupied volume 325. The first binding component 310 andthe second binding component 320 are attached to the retaining component300 at a separation distance Δs that causes the effective occupiedvolumes 315 and 325 to overlap, thereby creating an overlap volume ΔV.

Positioning of binding components on a detectable probe or affinityreagent may be determined, in whole or in part, by the structuralproperties of optional linkers that connect a binding component to aretaining component. Linkers may be used for various purposes, such asproviding separation between a retaining component and a bindingcomponent, positioning a binding component, providing attachment sitesfor other chemical entities, minimizing the likelihood of unwantedretaining component—binding component interactions, or generatingdesired chemical properties between a retaining component and bindingcomponent (e.g., hydrophobicity). A linker may include rigid orconformationally-constrained chemical groups (e.g., alkenes, alkynes,cyclic compounds). A linker may include flexible, dynamic, or moveablechemical groups (e.g., polyethylene glycol (PEG), polyethylene oxide(PEO), or alkane chains). A linker may include a polynucleotide that isnot configured to bind to other nucleic acids present in a method orapparatus where it is present. A linker including nucleic acids (e.g.,RNA, DNA, PNA) may include a polynucleotide that forms regions ofsecondary structure with itself (e.g., a hairpin, stem and/or loopstructure). A linker may provide additionally degrees of freedom formovement of a binding component attached to a retaining component.

FIGS. 4A-4D depict exemplary methods of controlling the volume that abinding component may occupy when attached to a retaining component by alinker. FIG. 4A depicts a schematic of a first binding component 410 anda second binding component 412 that are attached to a substantially flatretaining component surface 400 by linkers 420 and 422, respectively.The first binding component 410 and the second binding component 412have, respectively, static effective occupied volumes 415 and 417 (wherethe static effective occupied volumes are the maximum volumes occupiedby the binding components absent motion caused by the linker). Thelinkers 420 and 422 provide additional degrees of freedom for the firstbinding component 410 and the second binding component 412 to move,thereby creating dynamic effective occupied volumes 425 and 427,respectively. The first binding component 410 and the second bindingcomponent 412 are attached by linkers 420 and 422 at positions on theflat retaining component surface 400 that are separated by a distanceΔs₁ that creates a separation gap Δg₁ between the dynamic effectiveoccupied volumes 425 and 427, ensuring that the binding component cannotcontact or interact with each other. Alternatively, FIG. 4B depicts theuse of an intermediate chemical moiety to interrupt interactions betweentwo adjacent binding components. FIG. 4B depicts a schematic of a firstbinding component 410 and a second binding component 412, and anintermediate chemical moiety or blocking group 430 (e.g., PEG, PEO,alkane chains, dextran) that are attached to a substantially flatretaining component surface 400 by linkers 420, and 422, respectively.The first binding component 410, the second binding component 412, andthe blocking group 430 have, respectively, static effective occupiedvolumes 415, 417, and 435 (where the static effective occupied volumesare the maximum volumes occupied by the binding components absent motioncaused by the linker). The linkers 420 and 422 provide additionaldegrees of freedom for the first binding component 410 and the secondbinding component 412 to move, thereby creating dynamic effectiveoccupied volumes 425 and 427, respectively. The first binding component410 and the second binding component 412 are attached by linkers 420 and422 at positions on the flat retaining component surface 400 that areseparated by a distance Δs₂ and an effective separation gap Δg₂ iscreated between the dynamic effective occupied volumes 425 and 427 dueto hindrance by intermediate chemical moiety or blocking group 430(e.g., by steric repulsion). The use of an intermediate chemical moietyor blocking group 430 may decrease the necessary distance Δs₂ betweenbinding component on the substantially flat surface due to the blockingof inter-binding component interactions.

FIG. 4C depicts a schematic of minimizing binding component interactionsby altering the conformation of a retaining component. FIG. 4C depicts aschematic of a first binding component 410 and a second bindingcomponent 412 that are attached to a non-flat retaining componentsurface 450 by linkers 420 and 422, respectively. The first bindingcomponent 410 and the second binding component 412 have, respectively,static effective occupied volumes 415 and 417 (where the staticeffective occupied volumes are the maximum volumes occupied by thebinding components absent motion caused by the linker). The linkers 420and 422 provide additional degrees of freedom for the first bindingcomponent 410 and the second binding component 412 to move, therebycreating dynamic effective occupied volumes 425 and 427, respectively.The first binding component 410 and the second binding component 412 areattached by linkers 420 and 422 at positions on the non-flat retainingcomponent surface 450 that are separated by a distance Δs₃ that createsa separation gap Δg₃ between the dynamic effective occupied volumes 425and 427, ensuring that the binding components cannot contact or interactwith each other. Alternatively, FIG. 4D depicts the use of anintermediate chemical moiety to interrupt interactions between twoadjacent binding components. FIG. 4D depicts a schematic of a firstbinding component 410 and a second binding component 412, and anintermediate chemical moiety or blocking group 430 (e.g., PEG) that areattached to a non-flat retaining component surface 450 by linkers 420and 422, respectively. The first binding component 410, the secondbinding component 412, and the intermediate chemical moiety or blockinggroup 430 have, respectively, static effective occupied volumes 415,417, and 435 (where the static effective occupied volumes are themaximum volumes occupied by the binding components absent motion causedby the linker). The linkers 420 and 422 provide additional degrees offreedom for the first binding component 410 and the second bindingcomponent 412 to move, thereby creating dynamic effective occupiedvolumes 425 and 427, respectively. The first binding component 410 andthe second binding component 412 are attached by linkers 420 and 422 atpositions on the flat retaining component surface 450 that are separatedby a distance Δs₄ and an effective separation gap Δg₄ is created betweenthe dynamic effective occupied volumes 425 and 427 due to hindrance byintermediate chemical moiety 430 (e.g., by steric repulsion). The use ofan intermediate chemical moiety or blocking group 430 may decrease thenecessary distance Δs between binding components on the non-flat surfacedue to the blocking of inter-binding component interactions.

FIGS. 5A and 5B display alternative configurations for controlling therelative position and/or orientation of label components on a retainingcomponent including nucleic acids. FIG. 5A shows a short region ofhelical nucleic acids formed by hybridization between a continuousscaffold strand 510 and shorter staple oligonucleotides (e.g.,oligonucleotides 520 and 522). The oligonucleotides are occasionallyinterrupted by short strand breaks 525. The staple oligonucleotides 520and 522 further include fluorophores 530 and 532, respectively. Due tothe positions on oligonucleotides 520 and 522 where fluorophores 530 and532 are attached, the fluorophores 530 and 532 are positioned on thesame side of the retaining component with a separation distance Δs_(f1).The separation distance can be increased or decreased as necessary bychanging the attachment positions of the fluorophores 530 and 532, orchanging the oligonucleotides on which the fluorophores are attached.Alternatively, FIG. 5B shows a short region of helical nucleic acidsformed by hybridization between a continuous scaffold strand 510 andshorter staple oligonucleotides (e.g., oligonucleotides 520 and 522).The oligonucleotides are occasionally interrupted by short strand breaks525. The staple oligonucleotides 520 and 522 further includefluorophores 530 and 532, respectively. Due to the positions onoligonucleotides 520 and 522 where fluorophores 530 and 532 areattached, the fluorophores 530 and 532 are positioned on opposing sidesof the retaining component with a separation distance Δs_(f2). FIGS. 5Aand 5B demonstrate how similar separation distances between labelcomponents can be achieved by differing label orientations.

The detectable probes or affinity reagents of the present disclosure canbe configured to provide highly tunable platforms for displaying bindingcomponents and/or label components. The tunability of the detectableprobes or affinity reagents may manifest as the ability to customizeand/or optimize the avidity of the probe and/or the strength of thedetectable signal generated. The tunability may arise from thecustomizable retaining components that can be attached to one or morebinding components and/or attached to one or more label components atspecific locations on the probe.

The detectable probes or affinity reagents of the present disclosure maybe characterized as having a remarkably increased avidity. Withoutwishing to be bound by theory, the increased avidity of a detectableprobe or affinity reagent may derive from the presence of a plurality ofbinding components that, collectively increase the on-rate (e.g., asindicated by increased dissociation rate constant, k_(on)), decrease theoff-rate (e.g. as indicated by increased dissociation rate constant,k_(off)) of a detectable probe or affinity reagent from a bindingpartner, or decrease the likelihood that a probe will diffuse away froma binding partner before a binding component can re-bind to the bindingpartner. Surprisingly, it has been found that the detectable probe oraffinity reagent compositions of the present disclosure can display anaffinity for a binding partner that is at least an order of magnitudelarger than the affinity of any single binding component from theplurality of binding components attached to the probe, as characterizedby dissociation constants or binding on-rates or off-rates.

The tunable nature of detectable probes or affinity reagents may derive,in part, from the ability to customize the attachment of bindingcomponents to the retaining component of the probe. Several factors mayinfluence the strength of the avidity effect, including: 1) the totalnumber of binding components; 2) the location and/or orientation of thebinding components; 3) the areal or volumetric density of the bindingcomponents; 4) the affinities of the plurality of binding components; 5)the structure of the retaining component; and 6) the overall size of thedetectable probe or affinity reagent. Moreover, the design flexibilityof the described probe structures permits the inclusion of additionalcomponents that may increase avidity, such as pendant tails that mayweakly interact with other adjacent entities to temporarily localize thedetectable probe or affinity reagent near a binding partner.

The avidity and/or observability for a detectable probe or affinityreagent can be tuned by exploiting design flexibility of a retainingcomponent therein. A retaining component may be chosen if itprovides: 1) a 3-dimensional structure that provides a wide range ofpotential locations and orientations for binding component display; and2) the ability to attach binding components to the 3-dimensionalstructure at desired locations with high specificity. Of particularinterest are retaining components including nucleic acids that takeadvantage of the specificity of nucleic acid hybridization to createcomplex 3-dimensional structures with precisely located bindingpositions for binding components (and label components).

In some configurations, a nucleic acid structure (e.g., a DNA nanoballor a nucleic acid origami) may be utilized as a retaining component. Anucleic acid structure can yeild the advantage of providing a highdegree of spatial control over the location and orientation ofcomponents that are added to the retaining component. Nucleic acidstructures may typically have about 10 to 11 base pairs per turn of thehelical structure, meaning that each unique physical location within adouble-stranded nucleic acid has an associated angle of orientation.This property of nucleic acids facilitates the tunability of a nucleicacid-based retaining component structure by providing abundantvariations in position and orientation to customize the amount ofseparation between probe components, such as binding components and/orlabel components.

In other configurations, retaining components may have controlledspatial and/or orientation control of probe components by rationalcontrol or modification of retaining component structure and/orchemistry. Non-nucleic acid retaining components may be manipulated ormodified to produce retaining components with controlled or varied probecomponent location and/or orientation. In some cases, the location ororientation of probe components may be controlled by the shape orconformation of a non-nucleic acid particle, nanoparticle, or body. Forexample, shell-like structures or plate-like structures may providemultiple surfaces with varied properties that permit differentiallocation of binding components relative to label components. Moreover,non-nucleic acid retaining components may be provided with coatings orformed into composites in spatially controlled fashions, therebypermitting increased control over attachment locations and orientations.

FIGS. 6A-6F depict various simplified configurations of detectable probecompositions to demonstrate the flexibility of arrangement created by3-dimensional retaining component structures (e.g., DNA origami, carbonnanoparticles, silicon nanoparticles, etc.). FIG. 6A depicts top-downview of a rectangular or tile-shaped detectable probe. The probecontains a retaining component 610 that has a width and height as wellas a depth (not shown), creating a top face (shown), plus sides and abottom face (not shown). A plurality of binding components 620 isattached to the retaining component 610, the binding components being atspecific locations along the sides of the retaining component. Labelcomponents 630 are located at specific positions on the top face (andoptionally the bottom face) of the detectable probe. FIG. 6B depicts atop-down view of a rectangular or tile-shaped probe with a reversedattachment scheme from the probe shown in FIG. 6A. The probe contains aretaining component 610 that has a width and height as well as a depth(not shown), creating a top face (shown), plus sides and a bottom face(not shown). A plurality of binding components 620 are attached to theretaining component 610 at specific locations on the top face (andoptionally the bottom face) of the detectable probe. Label components630 are located at specific positions along the sides of the retainingcomponent. The probe configuration depicted in FIG. 6A may be preferablefor systems requiring a strong detection signal or a detection signalthat is distributed over a larger area or volume, due to the increasedlocations for labeling on the high-area top face. The probeconfiguration depicted in FIG. 6B may be preferable for increasing probeavidity of detectable probes or affinity reagents due to the potentialto increase the density of binding components on the high-area topsurface.

FIGS. 6C and 6D depict side-view configurations of rectangular ortile-shaped detectable probes, demonstrating the orientation of probecomponents relative to the thinner depth dimension of the probes. FIG.6C shows a detectable probe containing a retaining component 610 thathas a width and height (not shown) as well as a depth, creating sides,and a top and bottom face (not shown). A plurality of binding components620 is attached to the retaining component 610, the binding componentsattached at specific locations on the top face of the detectable probe.Label components 630 are located at specific positions on the bottomface of the detectable probe. FIG. 6D shows a detectable probecontaining a retaining component 610 that has a width and height (notshown) as well as a depth, creating sides, and a top and bottom face(not shown). A plurality of binding components 620 and label components630 is attached to the retaining component 610, the binding componentsattached at specific locations on the top and bottom faces of thedetectable probe. The probe configuration in FIG. 6C may be advantageousfor fluorescent detection system as the large probe may shield some orall of the binding partner from interacting with label components,thereby possibly decreasing or mitigating any quenching of the label bythe binding partner or its immediate environment. The probeconfiguration in FIG. 6D may be advantageous for maximizing the amountof binding component present for detectable probes or affinity reagentsby increasing the likelihood of contacting a detectable probe with abinding partner.

FIGS. 6E and 6F depict alternative retaining component geometries. FIG.6E depicts a detectable probe including a circular or sphericalconfiguration. The probe contains a retaining component 610 thatoptionally includes an internal structure that provides additionallocations for attachment of probe components. A plurality of bindingcomponents 620 are attached to the outer perimeter or surface of theretaining component 610. The internal region of the retaining component610 contains a plurality of label components 630. The configurationshown in FIG. 6E may increase the likelihood of the probe locating abinding partner due to the high coverage of binding components 620 onthe retaining component 610, and may further provide high detectablesignal due to the potential to concentrate a plurality of labelcomponents 630 in the internal space of the retaining component 610.FIG. 6F depicts a side view or cross-sectional view of a probe with aretaining component 610 that features angular offsets on one or morefaces. One side of the retaining component 610 contains attached bindingcomponents 620. The opposite side of the retaining component 610contains attached label components 630. The configuration shown in FIG.6F may be advantageous for increasing avidity due to the increasedvolumetric density of binding components 620 attached to the bottom faceof the retaining component 610. The more complex shape may increasecontact of binding components 620 with a binding partner and increaseresistance to diffusion of the probe away from a binding partner. Theskilled person will recognize that innumerable variations of thegeometries described in FIGS. 6A-6F may exist given the numerouspotential designs of the retaining component.

A retaining component may include a body (e.g. particle, nanoparticle,or microparticle) that is not primarily composed of nucleic acids. Aretaining component may be formed utilizing a retaining component thatis a fabricated or synthesized body, such as a silicon or silicananoparticle, a carbon nanoparticle, a cellulose nanobead, a PEGnanobead, a polymeric nanoparticle (e.g., polyacrylate particles,polystyrene-based particles, FluoSpheres™, etc.), or a quantum dot. Aparticle, nanoparticle, or body may include solid materials andshell-like materials (e.g., carbon nanospheres, silicon oxidenanoshells, iron oxide nanospheres, polymethylmethacrylate nanospheres,etc.). A retaining component including a particle, nanoparticle, or bodymay include distinct surfaces, such as plates or shells. In someconfigurations, distinct surfaces on a retaining component may beutilized to segregate components (e.g., binding components on a firstsurface, label components on a second surface). A retaining componentmay include a material that may be directly functionalized or modifiedto permit attachment of components (e.g., silanization of a silicon orsilicon dioxide nanoparticle). A retaining component may include acommercially-available particle, nanoparticle, or body. A retainingcomponent may be prepared by modifying a commercially-availableparticle, nanoparticle, or body. Methods and chemistries for modifyingstructures such as particles and nanoparticles are extensively describedin the art.

FIGS. 31A-31D illustrate various configurations for fashioning retainingcomponents from particles or nanoparticles. FIG. 31A depicts a solidspherical particle 3110 with a surface that is directly functionalizedwith a plurality of attachment sites 3120. The attachment sites may beconfigured to form covalent or non-covalent attachments to detectableprobe components, such as binding components and/or label components.FIG. 31B depicts a solid spherical particle 3110 that includes aheterogeneous plurality of first attachment sites 3120 and secondattachment sites 3125. In some configurations, there may be an equalnumber of first attachment sites 3120 and second attachment sites 3125.In other configurations, there may be differing numbers of firstattachment sites 3120 and second attachment sites 3125. The firstattachment sites 3120 and/or second attachment sites 3125 may beconfigured to form covalent or non-covalent attachments to detectableprobe components, such as binding components and/or label components.The solid spherical particles of FIGS. 31A and 31B may readily besubstituted with hollow particles. Hollow particles may be provided withattachment sites 3120 or 3125 on internal surfaces. In someconfigurations, a hollow particle may include a first plurality ofattachment sites 3120 on an external surface and a second plurality ofattachment sites 3125 on an internal surface to permit segregation ofdetectable probe components to differing surfaces.

FIGS. 31C and 31D illustrate retaining components formed from solidspherical particles including a surface coating, shell, or layer (e.g.,a polymer, hydrogel coating, or monolayer of functional groups). FIG.31C shows a solid spherical particle 3110 including a surface coating orlayer 3130. The coating or layer 3130 is provided with a plurality ofattachment sites 3120. The attachment sites may be configured to formcovalent or non-covalent attachments to detectable probe components,such as binding components and/or label components. FIG. 31D shows aspherical detectable particle 3115 (e.g., a FluoSphere™, a quantum dot)including a surface coating 3130. The coating or layer 3130 is providedwith a plurality of attachment sites 3120. The attachment sites may beconfigured to form covalent or non-covalent attachments to detectableprobe components, such as binding components and/or label components.The coating or layer may function as a scaffold that permits theattachment of binding components or other components to the particle,nanoparticle, or body. The skilled person will readily recognize thatthe described retaining components can readily be adapted tonon-spherical particles, nanoparticles, or bodies, such as nanotubes,plates, bowls, rods, and cones.

FIGS. 34A-34C illustrates various configurations of non-nucleic acidretaining components with spatially controlled or segregated probecomponents. FIG. 34A shows a spherical particle, nanoparticle, or body3410 that has spatially-segregated binding components 3420 and labelcomponents 3430. FIG. 34B shows a hollow or shell-like particle or body3412 that utilizes the hollow internal region of the particle or body3412 to segregate label components 3430 from externally attached bindingcomponent 3420. FIG. 34C shows a plate-like particle or body 3414 thathas two distinct surfaces with an approximately 180° angular offset. Theupper surface may be used to attach a plurality of label components 3430while the bottom surface may be used to attach a plurality of bindingcomponents 3420.

Two binding components may be attached to a retaining component in sucha fashion that they have an angular offset in terms of their relativepositions. For example, two binding components attached to a flat faceof a retaining component would have about 0 degree (°) angular offset.In another example, two binding components attached to opposite sides ofa cube-like retaining component would have about 180° angular offset. Anangular offset may be utilized to constrain contact or otherinteractions between adjacent binding components. Two binding componentsmay have a relative angular offset of at least about 0°, 5°, 10°, 15°,20°, 25°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 65°, 70°, 75°, 80°, 85°,90°, 95°, 100°, 105°, 110°, 115°, 120°, 125°, 130°, 135°, 140°, 145°,150°, 155°, 160°, 165°, 170°, 175°, or more. Alternatively oradditionally, two binding components may have a relative angular offsetof no more than about 180°, 175°, 170°, 165°, 160°, 155°, 150°, 145°,140°, 135°, 130°, 125°, 120°, 115°, 110°, 105°, 100°, 95°, 90°, 85°,80°, 75°, 70°, 65°, 60°, 55°, 50°, 45°, 40°, 35°, 30°, 25°, 20°, 15°,10°, 5°, or less.

A detectable probe or affinity reagent may be further modified toincrease the overall avidity of the probe. FIGS. 7A and 7B depict theuse of linkers to increase the spatial degrees of freedom for display ofbinding components. FIG. 7A depicts a detectable probe including aretaining component 710 with a plurality of attached binding components720 and a plurality of label components 730. A subset of the pluralityof binding components 720 is attached to the retaining component 710 bylinkers 740 (e.g., PEG, PEO, alkane chains, etc.) that permit the subsetof binding components 720 to extend away from the retaining component710. FIG. 7B depicts a similar probe configuration to FIG. 7A, howeverthe probe includes two differing species of binding components. Theprobe contains a retaining component 710 with a plurality of attachedlabel components 730. The probe further includes a first plurality ofattached binding components 720 (e.g., antibodies or antibody fragments)and a second plurality of attached binding components 722 (e.g.,aptamers) that are attached to the retaining component 710 by linkers740. Linkers may be advantageous for increasing probe avidity byfacilitating increased sensing of binding partners, epitopes, or targetmoieties over a larger volumetric region per unit of time. Moreover,linkers may provide flexibility or separation of components, and thismay speed up the binding of a probe to a binding partner, epitope, ortarget moiety, and slow the possible diffusion of the probe away fromthe binding partner, epitope, or target moiety.

The size of a detectable probe or affinity reagent may be configured tosuit an intended mode of use. The mode of use (e.g., polypeptidecharacterization, non-polypeptide characterization, therapeutics,diagnostics) may indicate the level of avidity and/or observability fora detectable probe or affinity reagent. A detectable probe or affinityreagent may be sized sufficiently to permit attachment of a sufficientnumber of binding components and/or label components for an intendedmode of use. In some configurations, the size of a detectable probe oraffinity reagent may refer to the approximate length, area, or volume ofthe retaining component. Components attached to retaining components(e.g., binding components, label components, linkers, blocking groups)may have increased degrees of spatial freedom that make characterizationof their length, areal size, or volumetric size more difficult.Retaining components may be configured to have a more regular or lessvariable size, making the retaining component size a viable proxy forsize of a detectable probe or affinity reagent.

A detectable probe, affinity reagent or retaining component thereof mayhave a characteristic length. A characteristic length may include amaximum, average or minimum length, for the width, height, radius,diameter, circumference, or other dimension. A detectable probe,affinity reagent or retaining component thereof may have acharacteristic length of at least about 5 nm, 10 nm, 15 nm, 20 nm, 25nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 120 nm, 140 nm, 160 nm, 180 nm,200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 600 nm, 700 nm,800 nm, 900 nm, 1000 nm, or more. Alternatively or additionally, adetectable probe, affinity reagent or retaining component thereof mayhave a characteristic length of no more than about 1000 nm, 900 nm, 800nm, 700 nm, 600 nm, 500 nm, 450 nm, 400 nm, 350 nm, 300 nm, 250 nm, 200nm, 180 nm, 160 nm, 140 nm, 120 nm, 100 nm, 95 nm, 90 nm, 85 nm, 80 nm,75 nm, 70 nm, 65 nm, 60 nm, 55 nm, 50 nm, 45 nm, 40 nm, 35 nm, 30 nm, 25nm, 20 nm, 15 nm, 10 nm, 5 nm, or less.

A detectable probe, affinity reagent or retaining component thereof mayhave a characteristic footprint (e.g. occupied area on a surface). Afootprint may constitute the area that a 2-dimensional projection of thedetectable probe, affinity reagent or retaining component thereof wouldcreate on a planar surface. A 2-dimensional projection may have aregular shape or an approximately regular shape, such as triangular,square, rectangular, circular, pentagonal, hexagonal, octagonal, orelliptic. FIGS. 2A-2B display examples of 2-dimensional projections forretaining components 210 or 215 with approximate shapes, with idealshapes shown as dashed lines (220 and 225, respectively). A detectableprobe, affinity reagent or retaining component thereof may have anoccupied area of at least about 25 nm², 100 nm², 500 nm², 1000 nm², 2000nm², 3000 nm², 4000 nm², 5000 nm², 5500 nm², 6000 nm², 6500 nm², 7000nm², 7500 nm², 8000 nm², 8500 nm², 9000 nm², 10000 nm², 15000 nm², 20000nm², 25000 nm², 50000 nm², 100000 nm², 1000000 nm², or more.Alternatively or additionally, a detectable probe, affinity reagent orretaining component thereof may have an occupied area of no more thanabout 1000000 nm², 100000 nm², 50000 nm², 25000 nm², 20000 nm², 15000nm², 10000 nm², 9000 nm², 8500 nm², 8000 nm², 7500 nm², 7000 nm², 6500nm², 6000 nm², 5500 nm², 5000 nm², 4000 nm², 3000 nm², 2000 nm², 1000nm², 500 nm², 100 nm², 25 nm², or less. The above ranges for occupiedarea can refer to an average for all faces of a detectable probe,affinity reagent or retaining component thereof; the smallest face ofthe detectable probe, affinity reagent or retaining component thereof;or the largest face of the detectable probe, affinity reagent orretaining component thereof, as desired.

A retaining component may be configured to have other components (e.g.binding components, label components or other retaining components)attached at a desired or optimal spacing. The spacing of bindingcomponents may be based on minimum spacing to reduce or eliminateunwanted or deleterious interactions (e.g., aptamer misfolding;fluorophore self-quenching). The spacing of binding components may bebased on maximum spacing to achieve desired characteristics, such asavidity or detectable signal strength. For components joined to aretaining component by a linker or other flexible method of attachment(i.e., components having additional degrees of freedom for motion), thespacing of attached components may be measured as the spacing betweenattachment sites on the retaining component. Two adjacent attachedcomponents (e.g., binding components, label components, blocking groups)may have a characteristic spacing of at least about 0.1 nm, 0.2 nm, 0.3nm, 0.4 nm, 0.5 nm, 0.6 nm, 0.7 nm, 0.8 nm, 0.9 nm, 1.0 nm, 1.1 nm, 1.2nm, 1.3 nm, 1.4 nm, 1.5 nm, 1.6 nm, 1.7 nm, 1.8 nm, 1.9 nm, 2 nm, 3 nm,4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm,15 nm, 16 nm, 17 nm, 18 nm, 19 nm, 20 nm, 21 nm, 22 nm, 23 nm, 24 nm, 25nm, 26 nm, 27 nm, 28 nm, 29 nm, 30 nm, 35 nm, 40 nm, or more.Alternatively or additionally, two adjacent attached components may havea characteristic spacing of no more than about 40 nm, 35 nm, 30 nm, 29nm, 28 nm, 27 nm, 26 nm, 25 nm, 24 nm, 23 nm, 22 nm, 21 nm, 20 nm, 19nm, 18 nm, 17 nm, 16 nm, 15 nm, 14 nm, 13 nm, 12 nm, 11 nm, 10 nm, 9 nm,8 nm, 7 nm, 6 nm, 5 nm, 4 nm, 3 nm, 2 nm, 1.9 nm, 1.8 nm, 1.7 nm, 1.6nm, 1.5 nm, 1.4 nm, 1.3 nm, 1.2 nm, 1.1 nm, 1.0 nm, 0.9 nm, 0.8 nm, 0.7nm, 0.6 nm, 0.5 nm, 0.4 nm, 0.3 nm, 0.2 nm, 0.1 nm, or less.

A detectable probe or affinity reagent may include a plurality ofbinding components that are attached to a surface or face of a retainingcomponent. The number of binding components displayed on a surface of adetectable probe or affinity reagent may be configured to increaseavidity or sufficiently space binding components to avoid unwantedinteractions. The number of binding components on a surface of adetectable probe or affinity reagent may be characterized as an averagenumber density (number per surface) or an area density (number perarea). Two differing surfaces or faces of a detectable probe or affinityreagent may have differing number or area densities of bindingcomponents. A detectable probe or affinity reagent may have an averagebinding component number density of at least about 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, or more per surface. Alternatively oradditionally, a detectable probe or affinity reagent may have an averagebinding component number density of no more than about 50, 49, 48, 47,46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29,28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, or less. A detectable probe or affinityreagent may have an average binding component area density of at leastabout 0.00001, 0.0001, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007,0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, or more per nm².Alternatively or additionally, a detectable probe or affinity reagentmay have an average binding component area density of no more than about1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06,0.05, 0.04, 0.03, 0.02, 0.01, 0.009, 0.008, 0.007, 0.006, 0.005, 0.004,0.003, 0.002, 0.001, 0.0001, 0.00001, or less per nm².

A detectable probe or affinity reagent may include a plurality of labelcomponents that are attached to a surface or face of a retainingcomponent. The number of label components displayed on a surface of adetectable probe or affinity reagent may be optimized to increaseobservability or sufficiently space label components to avoid unwantedinteractions. The number of label components on a surface of adetectable probe or affinity reagent may be characterized as an averagenumber density (number per surface) or an area density (number perarea). Two differing surfaces or faces of a detectable probe or affinityreagent may have differing number or area densities of label components.A detectable probe or affinity reagent may have an average labelcomponents number density of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, or more per surface. Alternatively or additionally,a detectable probe or affinity reagent may have an average labelcomponents number density of no more than about 50, 49, 48, 47, 46, 45,44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27,26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9,8, 7, 6, 5, 4, 3, 2, or less than 2 per surface. A detectable probe oraffinity reagent may have an average label components area density of atleast about 0.00001, 0.0001, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006,0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08,0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, or more per nm².Alternatively or additionally, a detectable probe or affinity reagentmay have an average label components area density of no more than about1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06,0.05, 0.04, 0.03, 0.02, 0.01, 0.009, 0.008, 0.007, 0.006, 0.005, 0.004,0.003, 0.002, 0.001, 0.0001, 0.00001, or less per nm².

A detectable probe or affinity reagent may include a heterogeneousmixture of binding components. A heterogeneous mixture of bindingcomponents may include mixtures of differing types and/or species ofbinding components. For example, a detectable probe or affinity reagentmay include a mixture of antibodies of differing affinity for a bindingpartner. In another example, a detectable probe or affinity reagent mayinclude a mixture of antibodies and aptamers that possess affinity forthe same binding partner. In another example, a detectable probe oraffinity reagent may include a mixture of antibodies and antibodyfragments with affinity for the same binding partner. A heterogeneousmixture of binding components may be advantageous for controlling theavidity of a detectable probe or affinity reagent. Mixtures of bindingcomponents with differing binding affinities may facilitate optimizationof the binding on-rate and/or binding off-rate for a detectable probe oraffinity reagent relative to a particular binding partner, epitope, ortarget moiety.

In some configurations, a detectable probe or affinity reagent mayinclude a plurality of first binding components with affinity for abinding partner, epitope, or target moiety, and a second plurality ofcompetitor binding components. Competitor binding components may becharacterized as having a decreased affinity (e.g., having an increasedbinding promiscuity, for example, binding a plurality of differentbinding partners, epitopes, or target moieties) with an increaseddissociation rate (e.g., a binding component that binds to many targetsbut easily dissociates from the targets). Without wishing to be bound bytheory, competitor binding components may be identified as bindingcomponents whose displacement by another binding component isenergetically and/or entropically favorable. For example, multiplesmall, low-affinity aptamers or mini-peptide binders may be easilydisplaced by a large, high-affinity antibody, thereby favoring antibodybinding due to the entropic increase of displacing multiple bindingcomponents. Competitor binding components may further increase theavidity of a detectable probe or affinity reagent by forming brief, weakinteractions with target moieties for which the first plurality ofbinding components lacks affinity. Such brief, weak interactions mayfacilitate increased duration of association between a detectable probeor affinity reagent and a binding partner, epitope, or target moiety.

A detectable probe or affinity reagent may be structurally stable undercertain environmental conditions. Of particular interest are detectableprobes or affinity reagents that are structurally stable underconditions that are intended to remove a bound probe from a bindingpartner. For example, a detectable probe or affinity reagent may bestable in the presence of heat or chemical compositions that interruptintermolecular interactions, e.g., surfactants or denaturants.Structural stability may refer to a detectable probe or affinity reagentmaintaining its full composition and, optionally, its shape orconformation, i.e., no loss of binding components, label components, orother components; no loss or degradation of components (e.g.,dehybridization of nucleic acids from a DNA origami retainingcomponent).

A detectable probe or affinity reagent may be structurally stable at agiven temperature. Temperature may vary during a process that utilizes adetectable probe or affinity reagent. For example, each step of binding,observing, and removing a detectable probe or affinity reagent may occurat a unique and/or optimal temperature. Moreover, a detectable probe oraffinity reagent may be structurally stable at a given storagetemperature. A detectable probe or affinity reagent may be structurallystable at a temperature of at least about −100° C., −90° C., −80° C.,−70° C., −60° C., −50° C., −40° C., −30° C., −20° C., −10° C., −5° C.,0° C., 4° C., 10° C., 15° C., 20° C., 25° C., 30° C., 35° C., 40° C.,45° C., 50° C., 55° C., 60° C., 65° C., 70° C., 75° C., 80° C., 90° C.,or higher. Alternatively or additionally, a detectable probe or affinityreagent may be structurally stable at a temperature of no more thanabout 90° C., 80° C., 75° C., 70° C., 65° C., 60° C., 55° C., 50° C.,45° C., 40° C., 35° C., 30° C., 25° C., 20° C., 15° C., 10° C., 4° C.,0° C., −10° C., −20° C., −30° C., −40° C., −50° C., −60° C., −70° C.,−80° C., −90° C., 100° C., or lower.

A detectable probe or affinity reagent may be structurally stable in thepresence of a particular solution or solvent. A detectable probe oraffinity reagent may be contacted with one or more solutions or solventsdepending upon the mode of use for the detectable probe or affinityreagent. Solutions or solvents may have varying compositions dependingupon the mode of use for a detectable probe or affinity reagent.Solutions or solvents may be used for processes such as probeformulation, probe storage, probe-partner binding, washing, rinsing,interaction detection, probe-partner separation, probe capture (e.g.,recovery of probes after utilization), and probe analysis (e.g.,post-process sequencing of nucleic acid barcodes). Solutions or solventsmay be formulated to maintain the stability of detectable probe oraffinity reagent structures. Solutions or solvents may be formulatedwith respect to chemical composition, pH, and ionic strength to ensurethe stability of detectable probe or affinity reagents. Detectableprobes or affinity reagents including nucleic acids may be present insolutions or solvents including magnesium salts to stabilize the nucleicacids.

A solution or solvent that is contacted with a detectable probe oraffinity reagent may include one or more detectable probes or affinityreagents in solution or suspension. A solution or solvent that iscontacted with a detectable probe or affinity reagent may be formulatedto be a homogeneous liquid medium. A solution or solvent that iscontacted with a detectable probe or affinity reagent may be formulatedto be a single-phase liquid medium. A solution or solvent that iscontacted with a detectable probe or affinity reagent may be formulatedto be a multi-phase liquid medium, such as an oil-in-water emulsion or awater-in-oil emulsion.

A solution or solvent that is contacted with a detectable probe oraffinity reagent may include a solvent species, pH buffering species, acationic species, an anionic species, a surfactant species, a denaturingspecies, or a combination thereof. A solvent species may include water,acetic acid, methanol, ethanol, n-propanol, isopropyl alcohol,n-butanol, formic acid, ammonia, propylene carbonate, nitromethane,dimethyl sulfoxide, acetonitrile, dimethylformamide, acetone, ethylacetate, tetrahydrofuran, dichloromethane, chloroform, carbontetrachloride, dimethyl ether, diethyl ether, 1-4, dioxane, toluene,benzene, cyclohexane, hexane, cyclopentane, pentane, or combinationsthereof. A solvent or solution may include a buffering speciesincluding, but not limited to, MES, Tris, Bis-tris, Bis-tris propane,ADA, ACES, PIPES, MOPSO, MOPS, BES, TES, HEPES, HEPBS, HEPPSO, DIPSO,MOBS, TAPSO, TAPS, TABS, POPSO, TEA, EPPS, Tricine, Gly-Gly, Bicine,AMPD, AMPSO, AMP, CHES, CAPSO, CAPS, and CABS. A solvent or solution mayinclude cationic species such as Na⁺, K⁺, Ag⁺, Cu⁺, NH₄ ⁺, Mg²⁺, Ca²⁺,Cu2 ⁺, Cd²⁺, Zn²⁺, Fe²⁺, Co²⁺, Ni²±, Cr²⁺, Mn²⁺, Ge²⁺, Sn²⁺, Al³⁺, Cr³⁺,Fe³⁺, Co³⁺, Ni³±, Ti³⁺, Mn³⁺, Si⁴⁺, V⁴⁺, Ti⁴⁺, Mn⁴⁺, Ge⁴⁺, Se⁴⁺, V⁵⁺,Mn⁵⁺, Mn⁶⁺, Se⁶⁺, and combinations thereof. A solvent or solution mayinclude anionic species such as F⁻, Cl⁻, Br⁻, ClO₃ ⁻, H₂PO₄ ⁻, HCO₃ ⁻,HSO₄ ⁻, OH⁻, I⁻, NO₃ ⁻, NO₂ ⁻, MnO₄ ⁻, SCN⁻, CO₃ ²⁻, CrO₄ ²⁻, Cr₂O₇ ²⁻,HPO₄ ²⁻, SO₄ ²⁻, SO₃ ²⁻, PO₄ ³⁻, and combinations thereof. A solvent orsolution may include a surfactant species including, but not limited to,stearic acid, lauric acid, oleic acid, sodium dodecyl sulfate, sodiumdodecyl benzene sulfonate, dodecylamine hydrochloride,hexadecyltrimethylammonium bromide, polyethylene oxide, nonylphenylethoxylates, Triton X, pentapropylene glycol monododecyl ether,octapropylene glycol monododecyl ether, pentaethylene glycol monododecylether, octaethylene glycol monododecyl ether, lauramide monoethylamine,lauramide diethylamine, octyl glucoside, decyl glucoside, laurylglucoside, Tween 20, Tween 80, n-dodecyl-β-D-maltoside, nonoxynol 9,glycerol monolaurate, polyethoxylated tallow amine, poloxamer,digitonin, zonyl FSO, 2,5-dimethyl-3-hexyne-2,5-diol, Igepal CA630,Aerosol-OT, triethylamine hydrochloride, cetrimonium bromide,benzethonium chloride, octenidine dihydrochloride, cetylpyridiniumchloride, adogen, dimethyldioctadecylammonium chloride, CHAPS, CHAPSO,cocamidopropyl betaine, amidosulfobetaine-16,lauryl-N,N-(dimethylammonio)butyrate,lauryl-N,N-(dimethyl)-glycinebetaine, hexadecyl phosphocholine,lauryldimethylamine N-oxide, lauryl-N,N-(dimethyl)-propanesulfonate,3-(1-pyridinio)-1-propanesulfonate,3-(4-tert-butyl-1-pyridinio)-1-propanesulfonate, N-laurylsarcosine, andcombinations thereof. A solvent or solution may include a denaturingspecies including, but not limited to, acetic acid, trichloroaceticacid, sulfosalicylic acid, sodium bicarbonate, ethanol, ethylenediaminetetraacetic acid (EDTA), urea, guanidinium chloride, lithiumperchlorate, sodium dodecyl sulfate, 2-mercaptoethanol, dithiothreitol,and tris(2-carboxyethyl)phosphine (TCEP).

A pH buffering species may be formulated in a solvent or solution in anyquantity. A pH buffering species may be present in a detectable probe oraffinity reagent solvent composition at a concentration of at leastabout 0.0001 M, 0.001 M, 0.01 M, 0.02 M, 0.03 M, 0.04 M, 0.05 M, 0.06 M,0.07 M, 0.08 M, 0.09 M, 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.6 M, 0.7 M,0.8 M, 0.9 M, 1 M, 1.1 M, 1.2 M, 1.3 M, 1.4 M, 1.5 M, 1.6 M, 1.7 M, 1.8M, 1.9 M, 2 M, 2.1 M, 2.2 M, 2.3 M, 2.4 M, 2.5 M, 2.6 M, 2.7 M, 2.8 M,2.9 M, 3 M, 3.1 M, 3.2 M, 3.3 M, 3.4 M, 3.5 M, 3.6 M, 3.7 M, 3.8 M, 3.9M, 4 M, 4.1 M, 4.2 M, 4.3 M, 4.4 M, 4.5 M, 4.6 M, 4.7 M, 4.8 M, 4.9 M, 5M, 5.1 M, 5.2 M, 5.3 M, 5.4 M, 5.5 M, 5.6 M, 5.7 M, 5.8 M, 5.9 M, 6 M, 7M, 8 M, 9 M or more. Alternatively or additionally, a pH bufferingspecies may be present in a solvent or solution at a concentration of nomore than about 10 M, 9 M, 8 M, 7 M, 6 M, 5.9 M, 5.8 M, 5.7 M, 5.6 M,5.5 M, 5.4 M, 5.3 M, 5.2 M, 5.1 M, 5.0 M, 4.9 M, 4.8 M, 4.7 M, 4.6 M,4.5 M, 4.4 M, 4.3 M, 4.2 M, 4.1 M, 4.0 M, 3.9 M, 3.8 M, 3.7 M, 3.6 M,3.5 M, 3.4 M, 3.3 M, 3.2 M, 3.1 M, 3.0 M, 2.9 M, 2.8 M, 2.7 M, 2.6 M,2.5 M, 2.4 M, 2.3 M, 2.2 M, 2.1 M, 2.0 M, 1.9 M, 1.8 M, 1.7 M, 1.6 M,1.5 M, 1.4 M, 1.3 M, 1.2 M, 1.1 M, 1.0 M, 0.9 M, 0.8 M, 0.7 M, 0.6 M,0.5 M, 0.4 M, 0.3 M, 0.2 M, 0.1 M, 0.09 M, 0.08 M, 0.07 M, 0.06 M, 0.05M, 0.04 M, 0.03 M, 0.02 M, 0.01 M, 0.001 M, 0.001 M, or less.

A cationic species may be formulated in a solvent or solution in anyquantity. A cationic species may be present in a detectable probe oraffinity reagent solvent composition at a concentration of at leastabout 0.0001 M, 0.001 M, 0.01 M, 0.02 M, 0.03 M, 0.04 M, 0.05 M, 0.06 M,0.07 M, 0.08 M, 0.09 M, 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.6 M, 0.7 M,0.8 M, 0.9 M, 1 M, 1.1 M, 1.2 M, 1.3 M, 1.4 M, 1.5 M, 1.6 M, 1.7 M, 1.8M, 1.9 M, 2 M, 2.1 M, 2.2 M, 2.3 M, 2.4 M, 2.5 M, 2.6 M, 2.7 M, 2.8 M,2.9 M, 3 M, 3.1 M, 3.2 M, 3.3 M, 3.4 M, 3.5 M, 3.6 M, 3.7 M, 3.8 M, 3.9M, 4 M, 4.1 M, 4.2 M, 4.3 M, 4.4 M, 4.5 M, 4.6 M, 4.7 M, 4.8 M, 4.9 M, 5M, 5.1 M, 5.2 M, 5.3 M, 5.4 M, 5.5 M, 5.6 M, 5.7 M, 5.8 M, 5.9 M, 6 M, 7M, 8 M, 9 M or more. Alternatively or additionally, a cationic speciesmay be present in a solvent or solution at a concentration of no morethan about 10 M, 9 M, 8 M, 7 M, 6 M, 5.9 M, 5.8 M, 5.7 M, 5.6 M, 5.5 M,5.4 M, 5.3 M, 5.2 M, 5.1 M, 5.0 M, 4.9 M, 4.8 M, 4.7 M, 4.6 M, 4.5 M,4.4 M, 4.3 M, 4.2 M, 4.1 M, 4.0 M, 3.9 M, 3.8 M, 3.7 M, 3.6 M, 3.5 M,3.4 M, 3.3 M, 3.2 M, 3.1 M, 3.0 M, 2.9 M, 2.8 M, 2.7 M, 2.6 M, 2.5 M,2.4 M, 2.3 M, 2.2 M, 2.1 M, 2.0 M, 1.9 M, 1.8 M, 1.7 M, 1.6 M, 1.5 M,1.4 M, 1.3 M, 1.2 M, 1.1 M, 1.0 M, 0.9 M, 0.8 M, 0.7 M, 0.6 M, 0.5 M,0.4 M, 0.3 M, 0.2 M, 0.1 M, 0.09 M, 0.08 M, 0.07 M, 0.06 M, 0.05 M, 0.04M, 0.03 M, 0.02 M, 0.01 M, 0.001 M, 0.001 M, or less.

An anionic species may be formulated in a solvent or solution in anyquantity. An anionic species may be present in a solvent or solution ata concentration of at least about 0.0001 M, 0.001 M, 0.01 M, 0.02 M,0.03 M, 0.04 M, 0.05 M, 0.06 M, 0.07 M, 0.08 M, 0.09 M, 0.1 M, 0.2 M,0.3 M, 0.4 M, 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, 1 M, 1.1 M, 1.2 M, 1.3M, 1.4 M, 1.5 M, 1.6 M, 1.7 M, 1.8 M, 1.9 M, 2 M, 2.1 M, 2.2 M, 2.3 M,2.4 M, 2.5 M, 2.6 M, 2.7 M, 2.8 M, 2.9 M, 3 M, 3.1 M, 3.2 M, 3.3 M, 3.4M, 3.5 M, 3.6 M, 3.7 M, 3.8 M, 3.9 M, 4 M, 4.1 M, 4.2 M, 4.3 M, 4.4 M,4.5 M, 4.6 M, 4.7 M, 4.8 M, 4.9 M, 5 M, 5.1 M, 5.2 M, 5.3 M, 5.4 M, 5.5M, 5.6 M, 5.7 M, 5.8 M, 5.9 M, 6 M, 7 M, 8 M, 9 M or more. Alternativelyor additionally, an anionic species may be present in a solvent orsolution at a concentration of no more than about 10 M, 9 M, 8 M, 7 M, 6M, 5.9 M, 5.8 M, 5.7 M, 5.6 M, 5.5 M, 5.4 M, 5.3 M, 5.2 M, 5.1 M, 5.0 M,4.9 M, 4.8 M, 4.7 M, 4.6 M, 4.5 M, 4.4 M, 4.3 M, 4.2 M, 4.1 M, 4.0 M,3.9 M, 3.8 M, 3.7 M, 3.6 M, 3.5 M, 3.4 M, 3.3 M, 3.2 M, 3.1 M, 3.0 M,2.9 M, 2.8 M, 2.7 M, 2.6 M, 2.5 M, 2.4 M, 2.3 M, 2.2 M, 2.1 M, 2.0 M,1.9 M, 1.8 M, 1.7 M, 1.6 M, 1.5 M, 1.4 M, 1.3 M, 1.2 M, 1.1 M, 1.0 M,0.9 M, 0.8 M, 0.7 M, 0.6 M, 0.5 M, 0.4 M, 0.3 M, 0.2 M, 0.1 M, 0.09 M,0.08 M, 0.07 M, 0.06 M, 0.05 M, 0.04 M, 0.03 M, 0.02 M, 0.01 M, 0.001 M,0.001 M, or less.

A surfactant species may be formulated in a solvent or solution in anyquantity. A surfactant species may be present in a solvent or solutionat a concentration of at least about 0.0001 M, 0.001 M, 0.01 M, 0.02 M,0.03 M, 0.04 M, 0.05 M, 0.06 M, 0.07 M, 0.08 M, 0.09 M, 0.1 M, 0.2 M,0.3 M, 0.4 M, 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, 1 M, 1.1 M, 1.2 M, 1.3M, 1.4 M, 1.5 M, 1.6 M, 1.7 M, 1.8 M, 1.9 M, 2 M, 2.1 M, 2.2 M, 2.3 M,2.4 M, 2.5 M, 2.6 M, 2.7 M, 2.8 M, 2.9 M, 3 M, 3.1 M, 3.2 M, 3.3 M, 3.4M, 3.5 M, 3.6 M, 3.7 M, 3.8 M, 3.9 M, 4 M, 4.1 M, 4.2 M, 4.3 M, 4.4 M,4.5 M, 4.6 M, 4.7 M, 4.8 M, 4.9 M, 5 M, 5.1 M, 5.2 M, 5.3 M, 5.4 M, 5.5M, 5.6 M, 5.7 M, 5.8 M, 5.9 M, 6 M, 7 M, 8 M, 9 M or more. Alternativelyor additionally, a surfactant species may be present in a solvent orsolution at a concentration of no more than about 10 M, 9 M, 8 M, 7 M, 6M, 5.9 M, 5.8 M, 5.7 M, 5.6 M, 5.5 M, 5.4 M, 5.3 M, 5.2 M, 5.1 M, 5.0 M,4.9 M, 4.8 M, 4.7 M, 4.6 M, 4.5 M, 4.4 M, 4.3 M, 4.2 M, 4.1 M, 4.0 M,3.9 M, 3.8 M, 3.7 M, 3.6 M, 3.5 M, 3.4 M, 3.3 M, 3.2 M, 3.1 M, 3.0 M,2.9 M, 2.8 M, 2.7 M, 2.6 M, 2.5 M, 2.4 M, 2.3 M, 2.2 M, 2.1 M, 2.0 M,1.9 M, 1.8 M, 1.7 M, 1.6 M, 1.5 M, 1.4 M, 1.3 M, 1.2 M, 1.1 M, 1.0 M,0.9 M, 0.8 M, 0.7 M, 0.6 M, 0.5 M, 0.4 M, 0.3 M, 0.2 M, 0.1 M, 0.09 M,0.08 M, 0.07 M, 0.06 M, 0.05 M, 0.04 M, 0.03 M, 0.02 M, 0.01 M, 0.001 M,0.001 M, or less.

A denaturing species may be formulated in a solvent or solution in anyquantity. A denaturing species may be present in a solvent or solutionat a concentration of at least about 0.0001 M, 0.001 M, 0.01 M, 0.02 M,0.03 M, 0.04 M, 0.05 M, 0.06 M, 0.07 M, 0.08 M, 0.09 M, 0.1 M, 0.2 M,0.3 M, 0.4 M, 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, 1 M, 1.1 M, 1.2 M, 1.3M, 1.4 M, 1.5 M, 1.6 M, 1.7 M, 1.8 M, 1.9 M, 2 M, 2.1 M, 2.2 M, 2.3 M,2.4 M, 2.5 M, 2.6 M, 2.7 M, 2.8 M, 2.9 M, 3 M, 3.1 M, 3.2 M, 3.3 M, 3.4M, 3.5 M, 3.6 M, 3.7 M, 3.8 M, 3.9 M, 4 M, 4.1 M, 4.2 M, 4.3 M, 4.4 M,4.5 M, 4.6 M, 4.7 M, 4.8 M, 4.9 M, 5 M, 5.1 M, 5.2 M, 5.3 M, 5.4 M, 5.5M, 5.6 M, 5.7 M, 5.8 M, 5.9 M, 6 M, 7 M, 8 M, 9 M or more. A denaturingspecies may be present in a solvent or solution at a concentration of nomore than about 10 M, 9 M, 8 M, 7 M, 6 M, 5.9 M, 5.8 M, 5.7 M, 5.6 M,5.5 M, 5.4 M, 5.3 M, 5.2 M, 5.1 M, 5.0 M, 4.9 M, 4.8 M, 4.7 M, 4.6 M,4.5 M, 4.4 M, 4.3 M, 4.2 M, 4.1 M, 4.0 M, 3.9 M, 3.8 M, 3.7 M, 3.6 M,3.5 M, 3.4 M, 3.3 M, 3.2 M, 3.1 M, 3.0 M, 2.9 M, 2.8 M, 2.7 M, 2.6 M,2.5 M, 2.4 M, 2.3 M, 2.2 M, 2.1 M, 2.0 M, 1.9 M, 1.8 M, 1.7 M, 1.6 M,1.5 M, 1.4 M, 1.3 M, 1.2 M, 1.1 M, 1.0 M, 0.9 M, 0.8 M, 0.7 M, 0.6 M,0.5 M, 0.4 M, 0.3 M, 0.2 M, 0.1 M, 0.09 M, 0.08 M, 0.07 M, 0.06 M, 0.05M, 0.04 M, 0.03 M, 0.02 M, 0.01 M, 0.001 M, 0.001 M, or less.

A solvent or solution may be formulated to have a pH at a value orwithin a range of values. A solvent or solution may have a pH of atleast about 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9,3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3,4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1,7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5,8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9,10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1,11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12.0, 12.1, 12.2, 12.3,12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13.0, 13.1, 13.2, 13.3, 13.4, 13.5,13.6, 13.7, 13.8, 13.9, 14.0 or more. Alternatively or additionally, asolvent or solution may have a pH of no more than about 14.0, 13.9,13.8, 13.7, 13.6, 13.5, 13.4, 13.3, 13.2, 13.1, 13.0, 12.9, 12.8, 12.7,12.6, 12.5, 12.4, 12.3, 12.2, 12.1, 12.0, 11.9, 11.8, 11.7, 11.6, 11.5,11.4, 11.3, 11.2, 11.1, 11.0, 10.9, 10.8, 10.7, 10.6, 10.5, 10.4, 10.3,10.2, 10.1, 10.0, 9.9, 9.8, 9.7, 9.6, 9.5, 9.4, 9.3, 9.2, 9.1, 9.0, 8.9,8.8, 8.7, 8.6, 8.5, 8.4, 8.3, 8.2, 8.1, 8.0, 7.9, 7.8, 7.7, 7.6, 7.5,7.4, 7.3, 7.2, 7.1, 7.0, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1,6.0, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1, 5.0, 4.9, 4.8, 4.7,4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3,3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9,1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5,0.4, 0.3, 0.2, 0.1, 0, or less.

A detectable probe or affinity reagent may include one or moreadditional modifying groups. Modifying groups may be included to alterthe chemical or physical properties of a detectable probe or affinityreagent. Modifying groups may be included on a detectable probe oraffinity reagent to alter a property such as hydrophobicity,hydrophilicity, amphiphilicity, electrical charge, magneticsusceptibility, or any other physical property. In some cases, modifyinggroups may be used to increase, decrease, or alter the solubility orsolution stability of a detectable probe or affinity reagent. In somecases, modifying groups may be used to maintain separation of detectableprobes or affinity reagents by mechanisms such as electrical repulsionor steric occlusion. In some cases, modifying groups may be used toincrease attraction between particles. For example, modifying groups mayform brief attractive interactions between detectable probes and/oraffinity reagents to create multi-probe complexes without causingaggregation or sedimentation of probes.

A surface of a retaining component may include one or more modifyinggroups. Modifying groups may be added to a surface to alter thecharacteristics of the surface while mediating an association between adetectable probe or affinity reagent and a surface or an interface. Forexample, hydrophobic modifying groups may be added to detectable probesor affinity reagents to cause probes to interact with oil droplets in anoil-in-water emulsion. Modifying groups may be attached covalently ornon-covalently. Modifying groups may be coupled to a retaining componentbefore, during, or after retaining component assembly. Surfacemodification groups may include electrically-charged moieties, magneticmoieties, steric moieties, amphipathic moieties, hydrophobic moieties,and hydrophilic moieties. Electrically-charged moieties may includefunctional groups that may carry an intrinsic positive or negativecharge, or may carry a charge under dissociating conditions (e.g.,carboxylic acids, nitrates, sulfones, phosphates, phosphonates, etc.).Magnetic moieties may include paramagnetic, diamagnetic, andferromagnetic particles such as nanoparticles (e.g., gadolinium,manganese, iron oxide, bismuth, gold, silver, cobalt nanoparticles,etc.). Steric moieties may include polymers and biopolymers (e.g., PEG,PEO, alkane chains, dextran, sheared nucleic acids). Amphipathicmoieties may include phospholipids (e.g., phosphatidic acid,phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine,phosphatidylinositol, phosphatidylinositol phosphate,phosphatidylinositol biphosphate, phosphatidylinositol triphosphate,ceramide phosphorylcholine, ceramide phophorylethanolamine, ceramidephosphoryllipid), glycolipids (e.g., glyceroglycolipids,sphingoglycolipids, rhamnolipids, etc.), and sterols (e.g., cholesterol,campesterol, sitosterol, stigmasterol, ergosterol, etc.). Hydrophobicmoieties may include steroids (e.g., cholesterol), saturated fatty acids(e.g., caprylic acid, capric acid, lauric acid, myristic acid, palmiticacid, stearic acid, arachidic acid, behenic acid, lignoceric acid,cerotic acid, etc.), and unsaturated fatty acids (e.g., myristoleicacid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid,vaccenic acid, linoleic acid, arachidonic acid, eicosapentaenoic acid,erucic acid, docosahexanenoic acid, etc.). Hydrophilic compounds mayinclude charged molecules and polar molecules (e.g., glycols,cyclodextrins, cellulose, polyacrylamides, etc.).

A surface of a detectable probe or affinity reagent may include one ormore modifying groups. A surface of a detectable probe or affinityreagent may include at least about 10, 50, 100, 500, 1000, 5000, 10000,50000, 100000, 50000, 1000000, or more modifying groups. Alternativelyor additionally, a surface of a detectable probe or affinity reagent mayinclude no more than about 1000000, 500000, 100000, 50000, 10000, 5000,1000, 500, 100, 50, 10, or fewer modifying groups.

A plurality of detectable probes, affinity reagents or both may beassembled into multi-probe complexes. A multi-probe complex can include,for example, a plurality of detectable probes, a plurality of affinityreagents, an affinity reagent and a detectable probe, an affinityreagent and a plurality of detectable probes, or a plurality of affinityreagents and a detectable probe. Multi-probe complexes may be preparedto further enhance the desirable properties of detectable probes oraffinity reagents, such as enhanced avidity and/or enhancedobservability. In some configurations, a multi-probe complex may includemultiple detectable probes, affinity reagents or both with similar oridentical configurations that are formed into a single complex. Amulti-probe complex including multiple similar or identical probes maybe formed to increase the overall brightness of probes when interactingwith a binding partner, or may provide an increased number of affinityreagents to increase the avidity over the avidity observed by a lonedetectable probe or affinity reagent. In other configurations, amulti-probe complex may include multiple detectable probes, affinityreagents or both with differing or dissimilar configurations that areattached into a single complex. A multi-probe complex may includemultiple detectable probes, affinity reagents or both with differing ordissimilar configurations may be formed to simultaneously bind tomultiple binding partners, epitopes, or target moieties, or increase thelikelihood of forming a binding interaction by increasing the number ofbinding partners, epitopes, or target moieties that the detectable probeor affinity reagent can recognize. A multi-probe complex may behave as aunivalent complex (e.g., configured to only bind a single species ofbinding partner, epitope, or target moiety) or a plurivalent complex(e.g., configured to bind to a variety of different binding partners,epitopes, or target moieties). Multi-probe complexes may be attached bya covalent or non-covalent interaction. A covalent bond between twodetectable probes, between two affinity reagents or between a probe andreagent may be formed by, for example a click reaction. A non-covalentinteraction between probes and/or reagents may be formed by, forexample, nucleic acid hybridization or a streptavidin-biotin coupling.

A multi-probe complex may be formed by coupling together two or moredetectable probes, affinity reagents or both. Multi-probe complexes maybe formed by direct attachment of detectable probe(s) and/or affinityreagent(s) (e.g., by nucleic acid hybridization, cross-linking, etc.).In some configurations, multi-probe complexes may be formed byattachment between one or more secondary retaining components, such asstructured nucleic acid particles or nanoparticles. Secondary retainingcomponents may be of particular interest if they provide tunablelocation and/or orientation of detectable probes or affinity reagents.In some configurations, a secondary retaining component may includeadditional components, such as modifying groups, binding components orlabel components. FIGS. 39A-39B show exemplary configurations ofmulti-probe complexes including a secondary retaining component. FIG.39A shows a secondary retaining component 3910 (e.g., a structurednucleic acid particle, a nanoparticle) including two or more attacheddetectable probes 3920 that are attached with an inward orientation. Thesecondary retaining component 3910 further includes a plurality ofattached label components 3930. FIG. 39B shows a secondary retainingcomponent including two or more separate particles 3910 (e.g., SNAPs,nanoparticles) that includes a plurality of detectable probes 3920 thatare coupled to form multiple pockets of inward oriented bindingcomponents. The secondary retaining component including the two or moreparticles 3910 further includes a plurality of label components 3930that are attached to the secondary retaining component. In otherconfigurations, a multi-probe complex may be formed by coupling orattaching multiple detectable probes or affinity reagents to anunstructured material or group, such as a polymer, metal, ceramic,semiconductor, glass, fiber, resin, or combination thereof. Anunstructured material may be amorphous, globular, porous, or somecombination thereof. An unstructured material may include a plurality ofattachment sites that permit attachment of a plurality of detectableprobes or affinity reagents.

FIGS. 35A-35C depict various configurations of multi-probe complexes.FIG. 35A depicts two identical or similar tile-shaped nucleic aciddetectable probes 3510 joined by a linking group 3520 (e.g., a covalentor non-covalent linking group). The detectable probes 3510 may have anaffinity for a single target or group of targets (e.g, a bindingpartner, epitope, or target moiety). FIG. 35B depicts a firsttile-shaped nucleic acid detectable probe with antibody bindingcomponents 3510 joined to a second tile-shaped detectable probe withaptamer binding components 3515 by a linking group 3520 (e.g., acovalent or non-covalent linking group). The detectable probes 3510 and3515 may have differing affinities, thereby allowing the detectableprobe to simultaneously bind more than one binding partner, epitope, ortarget moiety. FIG. 35C depicts a pair of non-nucleic acid-baseddetectable probes 3530 (e.g., particles, nanoparticles, etc.) that arejoined by a non-covalent attractive interaction, such as anelectrostatic or magnetic interaction. The non-nucleic acid-baseddetectable probes 3530 may include binding components with similar ordissimilar binding partners, epitopes, or target moieties.

In some configurations, multi-probe complexes may be configured to formcomplexes with controlled geometries. A controlled geometry may beemployed to orient binding components more favorably for increasedavidity. Multi-probe complexes may be configured to change shapes orconformations upon forming a binding interaction with a binding partner,epitope, or target moiety. Multi-probe complexes may be configured toresist changes to shape or conformation to increase contact or proximityof binding components to a binding partner, epitope, or target moiety.Individual probes or reagents included in a multi-probe complex may havemodifying groups that form interactions with other probes or reagents inthe multi-probe complex that affect the relative orientation of probeswithin the complex. FIG. 36A-36B depict configurations of detectableprobes with designed or structured geometries. FIG. 36A depicts twotile-shaped nucleic acid detectable probes 3610 that are joined by alinker 3620. Each probe 3610 include one or more modifying groups 3630(e.g., same electrical charge, same magnetic polarity, steric groups,etc.) that create a repulsion or hindrance to free motion of the linker3620. The modifying groups 3630 cause an inward orientation of theprobes, increasing the binding component density of the probe complex.FIG. 36B depicts two tile-shaped nucleic acid detectable probes 3610that are joined by a linker 3620. Each probe 3610 include one or moremodifying groups 3640 (e.g., hydrophobic groups, hydrophilic groups,etc.) that create an attraction between probes that creates an inwardorientation of the probes 3610, increasing the binding component densityof the probe complex. These configurations may resist deformation of thecomplex during a binding interaction with a binding partner.

A multi-complex may include two or more detectable probes and/oraffinity reagents that are attached into a single complex. A multi-probecomplex may include at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100, or more detectable probes and/or affinityreagents. Alternatively or additionally, a multi-probe complex mayinclude no more than about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50,45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7,6, 5, 4, 3, or fewer detectable probes and/or affinity reagents.

Affinity reagents or detectable probes described herein may include abinding component (also referred to as a binding component or probecomponent) that is capable of binding or associating with polypeptides,or other analytes, in a manner that allows one to interpret such bindingin order to facilitate identification of the polypeptide or otheranalyte with which it associates (or does not associate). Affinityreagents or detectable probes may also include a label component thatallows observation or detection of the binding or association of theaffinity reagent or detectable probe with a target analyte such as apolypeptide or other analyte. A label component can be separable from aprobe component, for example, when the label component is an exogenouslabel that is attached via a synthetic linker or attachment moiety.Alternatively, a label component can be an intrinsic characteristic of aprobe, such as a detectable mass, charge, or optical characteristic. Byidentifying which affinity reagents or detectable probes bind todifferent polypeptides or other analytes within a sample, one canidentify the presence, and potential quantity of such proteins orpolypeptides within that sample.

In some embodiments, an affinity reagent or detectable probe that isdirected towards identifying a target amino acid sequence may actuallyinclude a group of different components which are not differentiated ordistinguishable from each other as used in methods described herein. Inparticular, the different components that may be used to identify thesame target amino acid sequence may use the same label moiety toidentify the same target amino acid sequence. For example, an affinityreagent or detectable probe which binds a trimer amino acid sequence(AAA) regardless of flanking sequences may include either a singlebinding component which binds the trimer AAA sequence without any effectfrom flanking sequences, or a group of binding components (e.g. 400binding components), each of which binds to a different 5 amino acidepitope of the form αAAAβ, where α and β may be any amino acid. In anexample of the second case, the binding components may have a combinedeffect.

Binding and Avidity

For a single, monovalent affinity reagent, the strength of a bindinginteraction between the affinity reagent and a binding target (e.g., abinding partner, epitope, or target moiety) may be characterized by anaffinity. Affinity may be expressed quantitatively in the form of adissociation constant or an association constant. Without wishing to bebound by theory, a dissociation constant, K_(D), may arise from anequilibrium analysis of binding between an affinity reagent, A, and abinding partner, B, to form a bound complex AB. The rates of associationand disassociation between A and B depend upon the relativeconcentrations of A, B and AB, expressed as [A], [B], and [AB],respectively. The rate of association (or on-rate), r_(on), may beexpressed as:

r _(on) =k _(on) [A][B]  (1)

where k_(on) is the rate constant for binding. The rate ofdisassociation (or off-rate), r_(off), may be expressed as:

r _(off) =k _(off) [AB]  (2)

where k_(off) is the rate constant for dissociation. Based upon anequilibrium between the on-rate (1) and the off-rate (2), a dissociationconstant may be calculated as:

$\begin{matrix}{K_{D} = \frac{k_{off}}{k_{on}}} & (3)\end{matrix}$

For a given affinity reagent, smaller values of K_(D) indicate strongerbinding and higher values indicate weaker binding.

An apparent dissociation constant can be determined for a detectableprobe or affinity reagent that has a plurality of binding components.The apparent dissociation constant may be calculated analogously to thedissociation constant for a monovalent affinity reagent. Without wishingto be bound by theory, an apparent dissociation constant, K_(D,a), mayarise from an equilibrium analysis of binding between detectable probe(or affinity reagent), P, and a binding partner, B, to form a boundcomplex PB. The apparent rates of association and disassociation betweenP and B may depend upon the relative concentrations of P, B and PB,expressed as [P], [B], and [PB], respectively. The apparent rate ofassociation (or apparent on-rate), r_(on,a), may be expressed as:

r _(on,a) =k _(on,a) [P][B]  (4)

where k_(on,a) is the apparent rate constant for binding. The apparentrate of disassociation (or apparent off-rate), r_(off,a), may beexpressed as:

r _(off,a) =k _(off,a) [PB](5)

where k_(off,a) is the apparent rate constant for dissociation. Basedupon an equilibrium between the apparent on-rate (1) and the apparentoff-rate (2), an apparent dissociation constant may be calculated as:

$\begin{matrix}{K_{D,a} = \frac{k_{{off},a}}{k_{{on},a}}} & (6)\end{matrix}$

For a multivalent detectable probe or multivalent affinity reagent,smaller values of K_(D,a) indicate stronger binding and higher valuesindicate weaker binding.

Avidity of a detectable probe or affinity reagent may generally bedescribed as a decrease in the apparent dissociation constant for theprobe relative to the dissociation constants of its constitutive bindingcomponents. For example, for a probe including N binding components,each individual binding component may have an individual dissociationconstant as described by equations 1-3 above, e.g., {K_(D,1), K_(D,2), .. . K_(D,N)}. A detectable probe or affinity reagent having N bindingcomponents may be considered to have an increased avidity when theapparent dissociation constant for the detectable probe or affinityreagent as a whole, K_(D,a), is less than the respective dissociationconstants for each of the N binding components, e.g., K_(D,a)<{K_(D,1),K_(D,2), . . . K_(D,N)}. In the case where a detectable probe oraffinity reagent includes a plurality of N binding components ofidentical type and species whose dissociation constant is simply, K_(D)(i.e., K_(D,1)=K_(D,2)= . . . =K_(D,N)=K_(D)), then avidity may simplybe defined as K_(D,a)<K_(D). In the case where a detectable probe oraffinity reagent includes a plurality of N binding components ofdiffering types and/or species whose dissociation constants are{K_(D,1), K_(D,2), . . . K_(D,N)} and whose strongest binder isK_(D,min), then avidity may simply be defined as K_(D,a)<K_(D,min).

A detectable probe or affinity reagent of the present disclosure may bepreferred if it displays a K_(D,a) that is significantly smaller thanK_(D,min), for example K_(D,a)<K_(D,min) by about 1, 2, 3, 4, 5, 6, 7,8, 9, 10, or more than 10 orders of magnitude. A detectable probe oraffinity reagent may be selected for a particular binding target ifK_(D,a)<K_(D,min) by at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore than 10 orders of magnitude. Alternatively or additionally, adetectable probe or affinity reagent may be selected for a particularbinding target if K_(D,a)<K_(D,min) by no more than about 10, 9, 8, 7,6, 5, 4, 3, 2, 1 or less than 1 order of magnitude. A detectable probeor affinity reagent may be less favorable if K_(D,a)<K_(D,min) by toolarge an amount as it may indicate excessive binding as indicated by avery small off-rate.

The dissociation constant of a detectable probe, affinity reagent or abinding component thereof may be at least about 0.1 nM, 0.5 nM, 1 nM, 5nM, 10 nM, 15 nM, 20 nM, 25 nM, 30 nM, 35 nM, 40 nM, 45 nM, 50 nM, 55nM, 60 nM, 65 nM, 70 nM, 75 nM, 80 nM, 85 nM, 90 nM, 95 nM, 100 nM, 150nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 550 nM, 600nM, 650 nM, 700 nM, 750 nM, 800 nM, 850 nM, 900 nM, 950 nM, 1 μM, 2 μM,3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, or more. Alternativelyor additionally, the dissociation constant of a detectable probe,affinity reagent or a binding component thereof may be no more thanabout 10 μM, 9 μM, 8 μM, 7 μM, 6 μM, 5 μM, 4 μM, 3 μM, 2 μM, 1 μM, 950nM, 900 nM, 850 nM, 800 nM, 750 nM, 700 nM, 650 nM, 600 nM, 550 nM, 500nM, 450 nM, 400 nM, 350 nM, 300 nM, 250 nM, 200 nM, 150 nM, 100 nM, 95nM, 90 nM, 85 nM, 80 nM, 75 nM, 70 nM, 65 nM, 60 nM, 55 nM, 50 nM, 45nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 5 nM, 1 nM, 0.5 nM,0.1 nM, or less.

A detectable probe or affinity reagent may have an apparent or measureddissociation constant that is less than the dissociation constant for abinding component that is attached to the detectable probe or affinityreagent. A lower dissociation constant for a detectable probe oraffinity reagent may be attributed to an increased binding on-rate,increased icon, decreased binding off-rate, decreased k_(off), or acombination thereof. The decrease in an apparent or measureddissociation constant of a detectable probe or affinity reagent relativeto any binding component of the plurality of binding components attachedto the detectable probe or affinity reagent may be an N-fold decrease(i.e., K_(D,probe)=(1/N)K_(D,binding component)). The decrease in anapparent or measured dissociation rate constant of a detectable probe oraffinity reagent relative to any binding component of the plurality ofbinding components attached to the detectable probe or affinity reagentmay be at least about a 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 50-fold, 100-fold,250-fold, 500-fold, 1000-fold, or more. Alternatively or additionally,the decrease in an apparent or measured dissociation constant of adetectable probe or affinity reagent relative to any binding componentof the plurality of binding components attached to the detectable probeor affinity reagent may be no more than about a 1000-fold, 500-fold,250-fold, 100-fold, 50-fold, 25-fold, 20-fold, 15-fold, 10-fold, 9-fold,8-fold, 7-fold, 6-fold, 5-fold, 4-fold, 3-fold, 2-fold, or less.

In some cases, it may be useful to quantitatively describe avidity. Fora set of N binding components whose strongest binder for a bindingtarget has a dissociation constant K_(D,min), an avidity, A_(N), may bedefined as:

$\begin{matrix}{A_{N} = \frac{K_{D,\min}}{K_{D,a}}} & (7)\end{matrix}$

Where K_(D,a) is the apparent dissociation constant of a detectableprobe or affinity reagent including the set of N binding components. Insome configurations, a detectable probe or affinity reagent of thepresent disclosure may have an A_(N) that is greater than 1. The A_(N)of a detectable probe or affinity reagent may be at least about 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100, 125, 150, 200, 250, 300, 400, 500, 600, 700, 800,900, 1000, 2500, 5000, 10000, 25000, 50000, 100000, 1000000, 10000000,100000000, 1000000000, 10000000000, or more. Alternatively oradditionally, the A_(N) of a detectable probe or affinity reagent may beno more than about 10000000000, 1000000000, 100000000, 10000000,1000000, 100000, 50000, 25000, 10000, 5000, 2500, 1000, 900, 800, 700,600, 500, 400, 300, 250, 200, 150, 125, 100, 95, 90, 85, 80, 75, 70, 65,60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, orless.

Avidity may refer to the ability of an affinity reagent or detectableprobe to display an altered apparent affinity for a binding partner whenthe reagent or probe has multiple pathways for forming a bindinginteraction with the binding partner. For example, if a detectable probeor affinity reagent includes ten binding components, and each bindingcomponent has an affinity for a binding target X, the detectable probeor affinity reagent has ten possible pathways for forming a bindinginteraction with the target X. Avidity may be an amplified effect, inwhich the apparent affinity of a detectable probe or affinity reagentincluding a plurality of binding components for a binding partner,epitope, or target moiety may be stronger than the affinity of anybinding component of the plurality of binding components. For example, adetectable probe or affinity reagent with 10 binding components may havea measured dissociation constant of 50 nanomolar (nM) when the strongestbinding component attached to the detectable probe or affinity reagenthas a dissociation constant of only 800 nM.

Avidity may occur due to the tendency of a new binding interaction toform soon after a prior binding interaction is disrupted in the presenceof a plurality of binding components. Consequently, a bindinginteraction between a detectable probe (or affinity reagent) and abinding partner may be observed to occur over a longer time-scale thanan observed binding interaction between a single binding component andthe binding partner. Without wishing to be bound by theory, detectableprobe or affinity reagent binding may be tunable by variation of probedesign to alter the thermodynamic, kinetic, and/or mass transfercharacteristics of a probe. Avidity of a detectable probe or affinityreagent may be affected by factors that alter the likelihood of bindinginteractions, including: 1) binding component density on the probe; 2)total number of binding components on the probe; 3) types and/or speciesof binding components; 4) binding component binding thermodynamics; 6)binding component binding kinetics; 5) probe size and/or weight; 6)probe shape and/or conformation; and 7) secondary binding interactionmediators.

Depending upon the thermodynamic and kinetic binding behaviors of a poolof detectable probes or affinity reagents, increased concentration ofdetectable probe or affinity reagent may encourage the binding of adetectable probe or affinity reagent to a binding partner, epitope, ortarget moiety. The effective or targeted concentration of detectableprobes or affinity reagents utilized in a binding assay may becalculated with reference to the probes themselves, with reference tothe total number of binding components on a probe, or, in the case ofprobes with heterogeneous binding component mixtures, the number of aspecific species of binding component. For example, a solution providedat 0.1 mole/liter (M) of detectable probes or affinity reagents that arefabricated to contain 20 total binding components per probe at a 1:1antibody/aptamer ratio would have a 2 M total concentration of bindingcomponents or a 1 M concentration of aptamers or antibodies. Theeffective or targeted concentration of detectable probes or affinityreagents may be determined on a mass or molar basis. A detectable probe,affinity reagent or a binding component thereof may be provided for anintended purpose at a preferred concentration (e.g., a concentrationthat optimizes avidity).

A detectable probe, affinity reagent or a binding component thereof maybe provided at a concentration of at least about 1×10⁻¹⁵ M, 5×10⁻¹⁵ M,1×10⁻¹⁴ M, 5×10⁻¹⁴ M, 1×10⁻¹³ M, 5×10⁻¹³ M, 1×10⁻¹² M, 5×10⁻¹² M,1×10⁻¹¹ M, 5×10⁻¹¹ M, 1×10⁻¹⁰ M, 5×10⁻¹⁰ M, 1×10⁻⁹ M, 5×10⁻⁹ M, 1×10⁻⁸M, 5×10⁻⁸ M, 1×10⁻⁷ M, 5×10⁻⁷ M, 1×10⁻⁶ M, 5×10⁻⁶ M, 1×10⁻⁵ M, 5×10⁻⁵ M,1×10⁻⁴ M, 5×10⁻⁴ M, 1×10⁻³ M, 5×10⁻³ M, 1×10⁻² M, 5×10⁻² M, 1×10⁻¹ M,5×10 ⁻¹ M, 1 M, or higher. Alternatively or additionally, a detectableprobe, affinity reagent or a binding component thereof may be providedat a concentration of no more than about 1 M, 5×10⁻¹ M, 1×10⁻¹ M, 5×10⁻²M, 1×10⁻² M, 5×10⁻³ M, 1×10⁻³ M, 5×10⁻⁴ M, 1×10⁻⁴ M, 5×10⁻⁵ M, 1×10⁻⁵ M,5×10⁻⁶ M, 1×10⁻⁶ M, 5×10⁻⁷ M, 1×10⁻⁷ M, 5×10⁻⁸ M, 1×10⁻⁸ M, 5×10⁻⁹ M,1×10⁻⁹ M, 5×10⁻¹° M, 1×10⁻¹° M, 5×10⁻¹¹ M, 1×10⁻¹¹ M, 5×10⁻¹² M, 1×10⁻¹²M, 5×10⁻¹³ M, 1×10⁻¹³ M, 5×10⁻¹⁴ M, 1×10⁻¹⁴ M, 5×10⁻¹⁵ M, 1×10⁻¹⁵ M, orlower.

The size of a probe may have an effect on the avidity properties of adetectable probe or affinity reagent. Without wishing to be bound bytheory, the mass transfer properties of a detectable probe or affinityreagent may be affected by the size and/or weight of a probe. A masstransfer physical property (e.g., diffusion coefficient) of a detectableprobe or affinity reagent may have a scaling relationship for a masstransfer physical property that depends upon the size and/or weight ofthe probe. For example, a detectable probe or affinity reagent may havea single-component diffusion coefficient in water that scales with apower law relationship to the probe molecular weight (i.e., D˜MW⁻²). Alarger and/or heavier affinity reagent or detectable probe mayexperience a decrease in the diffusion rate in a solution or solvent. Adecreased diffusion rate of a detectable probe or affinity reagent maybe observed as a decrease in the apparent avidity of a detectable probeor affinity reagent due to an increased likelihood of a bindinginteraction occurring before a probe can diffuse away from a bindingpartner, epitope, or target moiety.

A probe may have a characterized molecular weight of at least about 1kDa, 10 kDa, 50 kDa, 100 kDa, 500 kDa, 1000 kDa, 1500 kDa, 2000 kDa,2500 kDa, 3000 kDa, 3500 kDa, 4000 kDa, 4500 kDa, 5000 kDa, 5500 kDa,6000 kDa, 6500 kDa, 7000 kDa, 7500 kDa, 8000 kDa, 8500 kDa, 9000 kDa,9500 kDa, 10000 kDa, or more. Alternatively or additionally, a probe mayhave a characterized molecular weight of no more than about 10000 kDa,9500 kDa, 9000 kDa, 8500 kDa, 8000 kDa, 7500 kDa, 7000 kDa, 6500 kDa,6000 kDa, 5500 kDa, 5000 kDa, 4500 kDa, 4000 kDa, 3500 kDa, 3000 kDa,2500 kDa, 2000 kDa, 1500 kDa, 1000 kDa, 500 kDa, 100 kDa, 50 kDa, 10kDa, 1 kDa, or less.

In some cases, the avidity of a detectable probe or affinity reagent maybe affected by a secondary binding interaction. A secondary bindinginteraction may be a weak or short-term interaction involving asecondary molecule that a detectable probe or affinity reagent has beendesigned to have. A secondary binding interaction may include a weak orshort-term interaction involving a secondary molecule that decreases theapparent rate of diffusion of the detectable probe or affinity reagentaway from a binding partner, epitope, or target moiety. A secondarybinding interaction may increase the likelihood of a detectable probe oraffinity reagent being observed in association with a binding partnerthat the detectable probe or affinity reagent for which the detectableprobe or affinity reagent has an affinity. In some configurations, asecondary molecule may include one or more ligands and/or moieties thatmay form a weak interaction with complementary ligands and/or moietieson a detectable probe or affinity reagent. For example, a secondarymolecule may have a nucleic acid sequence that is at least partiallycomplementary to a nucleic acid sequence in a detectable probe oraffinity reagent. As such, the secondary molecule and probe can beconfigured to form a weak interaction via hybridization of the twosequences. For example, weak interaction may result from imperfect ordiscontinuous hybridization such as interactions resulting from use ofhairpin or loop structures in the sequence of the detectable probe oraffinity reagent or secondary molecule, mismatched nucleotides inotherwise complementary regions of the detectable probe (or affinityreagent) and secondary molecule, or modified or unnatural nucleotides inthe detectable probe (or affinity reagent) or secondary molecule thatcreate weaker base pair binding. In other configurations, a secondarymolecule may include one or more ligands and/or moieties that may form aweak interaction with a binding partner. In some configurations, adetectable probe (or affinity reagent) or a secondary molecule mayinclude a plurality of molecules that are configured to form a weakbinding interaction.

In some configurations, a secondary molecule that interacts with adetectable probe or affinity reagent may be attached to a solid support.For example, the secondary molecule can be a SNAP, such as a nucleicacid origami or nucleic acid nanoball, that is attached to a solidsupport. The SNAP can optionally be attached to a binding partner forone or more binding components of a detectable probe or affinityreagent. Accordingly, a secondary molecule can mediate association of adetectable probe or affinity reagent with a solid support via binding ofone or more binding components to the binding partner and theassociation can be enhanced by a weak interaction between the secondarymolecule and the SNAP. In some configurations, a plurality of SNAPs isattached to a solid support in the form of an array. Each site in thearray can have a SNAP that is attached to a spatially resolved bindingpartner, and each of the binding partners can be recognized by one ormore detectable probes or affinity reagents. Exemplary SNAPs and solidsupports that can be usefully applied in a composition or method of thepresent disclosure are set forth in U.S. patent application Ser. No.17/062,405 (published as US Pat. App. Pub. No. 2021/0101930 A1) and WO2019/195633 A1, each of which is incorporated herein by reference.

FIG. 8 depicts a detectable probe 810 that is configured to form aninteraction with a binding partner 830 or an epitope or target moietywithin the binding partner 830. The detectable probe further includes apendant ligand 820 (e.g., a nucleic acid) that is configured to form aweak interaction with a complementary molecule 850 at an interactionregion 860. In some configurations, the secondary molecule 850 may bebound or associated with an anchoring group 840, the binding partner830, or a solid support 870. In some configurations, the interactionbetween the pendant ligand 820 and the complementary molecule 850 at theinteraction region 860 may be naturally unstable, facilitating theeventual dissociation of the detectable probe 810 from the bindingpartner 830. In other configurations, the interaction between thependant ligand 820 and the complementary molecule 850 at the interactionregion 860 may be configured to be disrupted (e.g., in the presence of adenaturant).

FIGS. 9A-9B depict possible configurations for forming weak interactionsbetween a detectable probe and a secondary molecule utilizing a pair ofcomplementary nucleic acids. FIG. 9A shows a first nucleic acid 910which may be coupled to a detectable probe or a secondary molecule and asecond nucleic acid 920 which may be coupled to the opposite molecule ofthe detectable probe-secondary molecule pair. The first nucleic acid 910is configured to hybridize to the second nucleic acid 920 in animperfect or discontinuous fashion, thereby forming a weak interactionregion 930. In some configurations, the imperfect or discontinuoushybridization may include hairpin or loop structures, mismatchednucleotides, or modified or unnatural nucleotides that create weakerbase pair binding. FIG. 9B shows a weak interaction between a detectableprobe and a secondary molecule that is formed between two nucleic acidsby a short hybridization region. A detectable probe 940 may include afirst nucleic acid 910 that is configured to hybridize with a secondnucleic acid 920. The first nucleic acid 910 and the second nucleic acid920 hybridize at a weak interaction region 930 which contains a limitednumber of bonded base pairs such that the base pair bonding has a degreeof reversibility or dissociation. The weak interaction region mayinclude a limited number of nucleotides such as, for example, no morethan about 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less thanabout 2 nucleotides. A weak base pairing interaction region may belocated at a terminal portion of a nucleic acid or in an internalportion of the nucleic acid sequence.

In some embodiments, the detectable probe further includes a partiallynon-hybridized nucleic acid having a single stranded region. In someembodiments, the single stranded region is bound to a complementarynucleic acid that is associated with a binding partner. In someembodiments, the single stranded region includes a primer sequence thatis configured to bind to a complementary primer sequence. In someembodiments, the primer sequence is configured to cause a toeholddisplacement reaction. In some embodiments, the primer sequence isconfigured to form a hairpin or loop structure when binding to thecomplementary primer sequence.

FIG. 10A-10C depict a toehold displacement strategy for forming a weakbinding interaction between a secondary molecule and a detectable probe.FIG. 10A shows the first step of a toehold displacement reaction. Adetectable probe 1010 including a first nucleic acid 1020 approaches asecond nucleic acid 1050 that is associated with a secondary molecule.The first nucleic molecule 1020 includes a hybridization sequencecontaining a toehold sequence 1030 and a priming sequence 1040. Thesecond nucleic acid 1050 includes a complementary hybridization sequenceincluding a complementary toehold sequence 1060 and a complementarypriming sequence 1070. The second nucleic acid 1050 may be hybridized toa toehold primer 1080 that only hybridizes to the complementary toeholdsequence 1060 of the hybridization sequence. The priming sequence 1040of the first nucleic acid 1020 may hybridize to the complementarypriming sequence 1070 of the second nucleic acid 1050, thereby formingan initial weak binding interaction between the detectable probe 1010and the secondary molecule. The weak binding interaction between thedetectable probe 1010 and the secondary molecule may have a degree ofreversibility or dissociation. FIG. 10B shows the second step of atoehold displacement reaction. The toehold sequence 1030 of the firstnucleic acid 1020 displaces the toehold primer 1080, allowing thetoehold sequence to complete hybridization with the complementarytoehold sequence 1060 of the second nucleic acid 1050. The continuedpresence of a toehold primer 1080 in the solution or solvent adjacent tothe weak binding interaction may cause reversal of the toeholddisplacement reaction by displacement of the first nucleic acid 1020from the second nucleic acid 1050 by the toehold primer 1080.

FIG. 10C depicts an alternative approach to forming a weak bindinginteraction between a detectable probe and a secondary molecule. Adetectable probe 1010 includes a first nucleic acid strand 1020 thatcontains a priming sequence 1040 that binds to a complementary primingsequence 1070 on a second nucleic acid strand 1050. The second nucleicacid strand 1050 also contains a complementary toehold sequence 1060that is not complementary to the sequence of the first nucleic acid1020. The detectable probe—secondary molecule complex may be contactedwith a solution or solvent that includes a toehold primer including atoehold sequence 1030 and a priming sequence 1040. The toehold primermay reversibly displace the first nucleic acid strand 1020 by disruptingthe base pair binding of the priming sequence 1040 with thecomplementary priming sequence 1070. In some configurations, theequilibrium between binding and displacement of a detectable probe maybe controlled by adjusting the concentration of one or more of thetoehold displacement reactants (e.g., toehold primers, detectableprobes).

The avidity of a detectable probe or affinity reagent may also beincreased by utilizing another binding molecule with a differingaffinity. In some configurations, an affinity reagent may include one ormore binding components, e.g., a retaining component attached to aplurality of binding components. In some configurations, an affinityreagent has a single binding component. In some configurations, abinding molecule is a detectable probe or affinity reagent. A bindingmolecule may have the property of binding to a binding partner, epitope,or target moiety with a lower affinity and/or avidity than a detectableprobe or affinity reagent. A binding molecule may be coupled to adetectable probe or affinity reagent such that the simultaneous bindingof the detectable probe or affinity reagent to the binding moleculecreates an apparent increase in the observed avidity or affinity of thedetectable probe or affinity reagent.

FIG. 11A-11C depict a system for increasing the avidity of a detectableprobe using a secondary binding interaction of a binding molecule. Asshown in FIG. 11A, a detectable probe 1110 is joined to a bindingmolecule 1130 (e.g., a second detectable probe) by a linker 1190 (e.g.,a covalent heterobifunctional linker, hybridized nucleic acids, etc.).The linker can be flexible, for example, allowing the detectable probe1110 to interact directly with the binding molecule 1130. Alternatively,the linker can be relatively rigid, for example, constraining thedetectable probe 1110 from directly interacting with the bindingmolecule 1130. The detectable probe 1110 includes a first plurality ofbinding components 1120, and the binding molecule 1130 includes a secondplurality of binding components 1140. A composition including thecomplex formed by the joining of the detectable probe 1110 and thebinding molecule 1130 is contacted with a binding partner 1150 thatcontains an epitope or target moiety 1160, and a secondary binding site1170. As shown in FIG. 11B, a binding interaction may be initiated bythe initial binding of a binding component of the first plurality ofbinding components 1120 with the epitope or target moiety 1160. At thetime of initial binding, the binding molecule may not yet have formed asecondary interaction with the binding partner 1150. As shown in FIG.11C, the binding process may be complete when a binding component of thesecond plurality of binding components 1140 forms a secondary bindinginteraction with the secondary binding site 1170, thereby binding thedetectable probe 1110 and the binding molecule 1130 to the bindingpartner 1150. The weak secondary binding interaction between the bindingmolecule 1130 and the secondary binding target 1170 may increase thelikelihood that the detectable probe 1110 will be observed as beingbound to the binding partner 1150. The skilled person will readilyrecognize that the binding order of a binding complex may be reversed,e.g., a binding molecule 1130 binds a secondary binding site 1170 first,followed by binding of a detectable probe 1110 to the epitope or targetmoiety 1160.

FIGS. 11D-11E depict an alternative system for increasing the avidity ofa detectable probe using a secondary binding interaction of a bindingmolecule. As shown in FIG. 11D, a detectable probe 1110 including afirst plurality of binding components 1120 and a first linking group1191 including a linking region 1192 is bound to an epitope or targetmoiety 1160 of a binding partner 1150. An unbound binding molecule 1130including a second plurality of binding components 1140 and a secondlinking group 1193 including a complementary linking region 1194 iscontacted with the binding partner 1150. As shown in FIG. 11E, a bindingcomponent of the plurality of binding components 1140 binds the bindingmolecule 1130 to a secondary binding site 1170 of the binding partner,thereby forming a weak secondary binding interaction. After the bindingof the binding molecule 1130, the linking region 1192 and thecomplementary linking region 1194 can become joined, thereby forming alinker between the detectable probe 1110 and the binding molecule 1130.The weak secondary binding interaction between the binding molecule 1130and the secondary binding target 1170 may increase the likelihood thatthe detectable probe 1110 will be observed as being bound to the bindingpartner 1150. The skilled person will readily recognize that the bindingorder of a binding complex may be reversed, e.g., a binding molecule1130 binds a secondary binding site 1170 first, followed by binding of adetectable probe 1110 to the epitope or target moiety 1160. The firstlinking group 1191 and the second linking group 1193 may be configuredto form a covalent or non-covalent interaction. For example, a firstlinking group 1191 may include a nucleic acid linking region 1192 and asecond linking group 1193 may include a complementary nucleic acidlinking region 1194 that is configured to hybridize to the nucleic acidlinking region 1192. In another example, a linking region 1192 mayinclude a functional group (e.g., a click reaction group) that isconfigured to chemically bond to a functional group in the complementarylinking region 1194 (e.g., by a click reaction).

In many cases, an affinity reagent, detectable probe or bindingcomponent thereof may have a non-random probability of binding toselected binding partners (e.g. polypeptide molecules or amino acidepitopes within larger polypeptide structures), meaning that it displayssome level of heightened affinity for a given epitope or set of epitopesover others, also termed “specificity”. In many cases, specificity maynot be binary, meaning that an affinity reagent or detectable probe maysometimes not appear to bind to a given epitope for which it hasdisplayed heightened specificity, and sometimes it may appear to bind toan epitope for which it has no demonstrated specificity. In many cases,whether a given affinity reagent or detectable probe is specific for agiven epitope or binding partner as described above, will be correlatedwith the probability of that affinity reagent or detectable probebinding to the given epitope or binding partner under the conditions ofthe binding assay. For example, an affinity reagent or detectable probecan be considered specific for a particular epitope or binding partnerif the probability of binding is greater than a probability threshold.The probability threshold can be known prior to performing the bindingassay (i.e. the threshold is predetermined) or it can determined fromthe binding assay itself (i.e. the threshold is empirically determined).In some cases, the probability threshold that is correlated withspecific binding may be greater than 50%, 60%, 70%, 80%, 90%, 95%, 99%or more. In some cases, the probability threshold that is correlatedwith specific binding may be greater than 1%, 5%, 10%, 20%, 30%, or 40%.Conversely, the probability that a given affinity reagent or detectableprobe does not bind a given epitope or binding partner under a given setof circumstances (e.g., providing a false negative binding result) may,in some cases, range from less than 1% to greater than 99%, meaning thatthere may be a significant likelihood of affinity reagent or detectableprobes not binding to their complementary epitope or polypeptide. Whilein some cases, the probability threshold that is correlated withnon-specificity or non-binding will be less than 50%, 40%, 30%, 20%,10%, 5%, or less. In some cases, the probability threshold that iscorrelated with non-specificity or non-binding may be less than 60%,70%, 80%, 90%, or 95%.

In some embodiments, a detectable probe or affinity reagent may have oneof more of the following properties: may specifically bind to aparticular amino acid species and may not bind to more than nineteenother amino acid species. For example, a detectable probe or affinityreagent may bind to one of the 20 essential amino acids but not theother 19 essential amino acids. A detectable probe or affinity reagentmay bind at least 10% of sequences of the form αXβ, wherein X is thedesired epitope and α and β are any amino acid residues. In some cases,the detectable probe or affinity reagent may bind at least 0.25%, 0.5%,0.75%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,20%, 30%, 40%, 50%, 75%, or 90% of sequences of the form αXβ. In somecases, a detectable probe or affinity reagent may bind to at least 10%of sequences of the form αXβ with a K_(D) value at least 10 fold lowerthan an average K_(D) value of the detectable probe or affinity reagentfor a pool of random sequences. In some cases, the detectable probe oraffinity reagent may bind to at least 10% of sequences of the form αXβwith a K_(D) value at least 100 fold lower than an average K_(D) valueof the detectable probe or affinity reagent for a pool of randomsequences. In some cases, the detectable probe or affinity reagent maybind to at least 10% of sequences of the form αXβ with a K_(D) value atleast 1000 fold lower than an average K_(D) value of the detectableprobe or affinity reagent for a pool of random sequences.

In some embodiments, a detectable probe or affinity reagent may have oneor more of the following properties: may specifically bind a desiredepitope having a particular three amino acid sequence, may not bind anyother three amino acid sequence, and may bind the desired epitope withsubstantially similar affinity regardless of flanking sequencesurrounding the desired epitope. In some cases, the detectable probe oraffinity reagent may have some, more, or all of these properties. Insome cases, the detectable probe or affinity reagent may not bind asubset of the epitope. Another aspect of the present disclosure providesa detectable probe or affinity reagent which may preferentially bind aknown set of three amino acid epitopes for which the preference forthese epitopes relative to other epitopes, and subject to flanking aminoacid residues, has been determined.

In some cases, a detectable probe or affinity reagent may include aswitchable aptamer which binds to between 5% and 10% of all proteins inthe human proteome. In some cases, the switchable aptamer may includetwo or more fluorescent moieties. A switchable aptamer may be a bindingcomponent of a detectable probe or affinity reagent of the presentdisclosure.

In some cases, a detectable probe or affinity reagent is configured tobind to a given sequence with a K_(D) value for the detectable probe oraffinity reagent and the given sequence that is at least 10, 100, or1000 fold lower than an average K_(D) value of the detectable probe oraffinity reagent for a pool of proteins having random sequences.

In some cases, a detectable probe or affinity reagent may have anability to bind a desired epitope that is in one or more structuralcontext. In some embodiments, a detectable probe or affinity reagent maybind a desired epitope when a polypeptide having the epitope is in adenatured context, in a native context, or both. In some embodiments, adetectable probe or affinity reagent may have an ability to bind adesired epitope in a polypeptide within a folded or unfolded context. Insome embodiments, polypeptides that have been denatured may contain orgenerate one or more microfold regions within the polypeptides.Detectable probes or affinity reagents may be designed to bind a desiredepitope in a polypeptide that has been allowed to fold after having beendenatured. The resulting polypeptide may be renatured to its activefolded state, partially refolded, or folded into a different state. Adetectable probe or affinity reagent may bind to an epitope in a foldedor unfolded region of a polypeptide that has been denatured and/orallowed to fold from the denatured state.

In some embodiments, a detectable probe or affinity reagent thatrecognizes a desired epitope sequence (e.g. AAA) may bind equally well,or nearly equally well, to all polypeptides containing the epitopesequence. In some cases, detectable probes or affinity reagents may bindto a desired epitope with differing affinities according to differencesin the sequence context of the epitope. In some cases, detectable probesor affinity reagents may bind several different epitopes regardless ofsequence context. In some cases, detectable probes or affinity reagentsof this disclosure may bind several different epitopes with differentaffinities depending on sequence context. Detectable probes or affinityreagents with such properties may be identified by a combination ofscreening methods to determine the binding characteristics of thedetectable probe or affinity reagent. For example, a detectable probe oraffinity reagent may be screened for the ability to bind to a set ofpolypeptide sequences that differ from one another except for a commoncore sequence (e.g. an amino acid epitope of the form αAAAβ, where AAAis the common core sequence and α and β may be any amino acid).

In some cases, the desired epitope of a detectable probe or affinityreagent may be a peptide sequence. In some cases, several differentepitopes may be desired. In this case a detectable probe or affinityreagent may be selected which binds the plurality of desired epitopes.In some cases, the desired epitope or epitopes may be referred to as X.In some cases, the epitope includes a non-contiguous amino acidsequence. For example, an epitope may include a specified amino acidevery second, third or fourth amino acid residue in a region of theprimary sequence of a polypeptide. An epitope may include a sequence of3 amino acids in which two specified amino acids are separated by avariable amino acid (e.g. AαA, where A is alanine and α is any aminoacid), a sequence of 4 amino acids in which two specified amino acidsare separated by two variable amino acids (e.g. AαβA where A is alanineand α and β are any amino acid), a sequence of 5 amino acids in whichtwo specified amino acids are separated by three variable amino acids,etc. In some embodiments, an epitope can include a sequence of two ormore of the non-contiguous epitopes. In another example, an epitope mayinclude several amino acid residues that are located proximally to eachother in a protein secondary or tertiary structure even though theresidues are not proximal in the protein sequence (i.e. not proximal inthe primary structure of the protein). In some cases, the epitopeincludes a contiguous sequence of specified amino acids. In someembodiments, the desired epitope, X, is a short amino acid sequence, of2, 3, 4, 5, 6 or 7 amino acids. In some cases, X includes severaldifferent short amino acid sequences. In some embodiments, the desiredepitope, X, is a three amino acid sequence, X₁X₂X₃. Detectable probes oraffinity reagents which bind this desired epitope in a variety ofsequence contexts may be identified by screening for binding to targetpolypeptides including the desired epitope.

The target may include a plurality of polypeptides which include thedesired sequence, X. The plurality may have any of a variety ofconfigurations such as a pool of polypeptides in solution-phase, anarray of polypeptides on a solid support, in a vessel, on a solidsupport, among a collection of vessels each containing one or more ofthe polypeptides in the plurality, etc. In some cases, the target is aplurality of polypeptides all of sequence X. In some embodiments thetarget may include a plurality of polypeptides of sequence αXβ, whereinX is the desired epitope and α and β may be any sequence of zero, one,or more than one amino acids. For example, if the desired epitope, X, isAAA, then examples of the sequences which may be found in the targetpolypeptides may include: AAAAA (SEQ ID NO: 1), AAAAC (SEQ ID NO: 2),CAAAA (SEQ ID NO: 3), CAAAC (SEQ ID NO: 4), and CAAAD (SEQ ID NO: 5). Insome cases, α and β may each be any single amino acid. The amino acidfor a may be the same as, or different from, the amino acid for (3. Insome cases, at least one of α and β may be 2, 3, 4, 5, 6, 7, 8, 9, 10,or more amino acids. The sequence for a may be the same as, or differentfrom, the sequence for β. In some cases, at least one of α and β mayinclude a linker or spacer. The linkers or spacers may be any linkers orspacers set forth herein or known in the art. In some cases, the linkerhas a peptide backbone, for example being an amino acid linker. In somecases, the linker is a polyethylene glycol (PEG) or a PEG polymer chain.The PEG chain may consist of 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 34, 36, 38, 40, 42, 44, 46, 48, 50 or moreethylene glycol monomers. In some cases, the linker may be a carbonchain. The polypeptides may also include an N terminal or C terminalmodification, for example, capping. In some cases, the polypeptides maybe modified to remove a charge, for example, terminal amidation(C-terminus) or acetylation (N-terminus). In some cases, the αXβ peptidemay contain non-naturally occurring amino acids. In some cases, the αXβpeptide may be modified with a linker and a functional group. Forexample, the molecule may be of the structure F-L-αXβ, where F is afunctional group and L is a linker. In other cases, the molecule may beof the structure αXβ-L-F, where F is a functional group and L is alinker. Functional group F can optionally be capable of forming acovalent bond with a reactive moiety or binding to a receptor. In somecases, α and β may each be glycine, or may each be one or more glycineresidues. In some embodiments, amino acid residues may be modified toalter their aptagenicity. For example, residues may be altered by addinga positive charge; adding a negative charge; adding a hydrophobic group;modified so as to add a sugar; or other modifications so as to increasechemical diversity.

Polypeptides may be synthesized using any method known in the art.Several commercial platforms exist for polypeptide synthesis, such asthe MultiPep RSi synthesizer (Intavis, Germany). Polypeptides may besynthesized using liquid phase or solid phase methods. Synthesizedpolypeptides may be verified using any known method for polypeptideanalysis. For example, polypeptides may be verified using Massspectrometry, Matrix Assisted Laser Desorption/Ionization Time of FlightMass spectrometry (MALDI-TOF), Matrix Assisted LaserDesorption/Ionization, AMS (Accelerator Mass Spectrometry), GasChromatography-MS, Liquid Chromatography-MS, Inductively CoupledPlasma-Mass spectrometry (ICP-MS), Isotope Ratio Mass Spectrometry(IRMS), Ion Mobility Spectrometry-MS, Tandem MS, Thermal Ionization-MassSpectrometry (TIMS), or Spark Source Mass Spectrometry (SSMS).Concentration of the synthesized peptides may also be assessed byspectroscopy.

FIG. 48 illustrates an immobilized target for selection orcharacterization of detectable probes or affinity reagents, along withan exemplary list of polypeptides which include the target. In theexample of FIG. 48 the desired epitope is AAA, and the polypeptides ofthe target include sequences αAAAβ, wherein α and β are each a singleamino acid. In this example the target includes 400 differentpolypeptides, representing each possible sequence of αAAAβ, wherein αand β are each a single amino acid.

In this way, for any given 3-mer epitope a target including a pool of5-mers may contain 400 different sequences (20 possibilities for α and20 possibilities for β, where each of α and β are a single amino acid).In some cases, the target may include a pool of polypeptides longer than5 amino acids in which each or both of α and β may include two or moreamino acids. In some cases, one of α and β may include zero amino acids,and the other of α or β may include one or more amino acids. In somecases, the target may include a polypeptide of sequence X withoutadditional amino acids.

In some cases, the target sequence X may be embedded in a longersequence. For example the target sequence X may be a core sequence offewer than 15 amino acids that is embedded in a 15-mer polypeptidemolecule. The target sequence X may be embedded at any position withinthe 15-mer, for example in the case of a three amino acid targetsequence X, the target sequence X may begin at position 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, or 13 of the 15-mer. Polypeptides includingembedded target sequences may be synthesized in solution, or may besynthesized on a chip, such as for example a PEPperPRINT chip or otherpeptide array. In some embodiments, polypeptides including embeddedtarget sequences may be bound or synthesized onto a single moleculeprotein array. The longer sequence may be selected to form a secondarystructure, or to lack secondary structure. Examples of such secondarystructures include alpha helices, beta sheets, proline bends, turns,loops, and cysteine bridges. In some cases, the longer sequence mayinclude non-naturally occurring amino acids, or other groups.

An initial selection step may include screening a library of affinityreagents or detectable probes against a target which includes a desiredepitope. The affinity reagent or detectable probe or probe library mayinclude DNA, RNA, or peptide aptamers with random sequences, or withsequences similar to those of known protein binding aptamers.

In some cases, the library may be an aptamer library. In some cases, anaptamer library may be a commercial library. In some cases, an aptamerlibrary may be available from an institute, university, academic center,or research center. In some cases, a library may include a library ofaptamers attached to beads or other particles. In some cases, an aptamerlibrary may be generated from a library of known sequences, or fromrandom sequences. In some cases, an aptamer library may include aptamerswith particular structures, such as, for example, a stem loop library.In some cases, the aptamer library may include switchableaptamers—aptamers which can be switched between two conformations. Forexample, an aptamer may form a first conformation in the presence of ametal ion cofactor and a second conformation in the absence of thecofactor. Accordingly, adding a chelating agent such as EDTA, or EGTA,sequesters the metal ions and causes the aptamer to adapt a differentconformation. Other factors that may be used to induce aptamer switchinginclude light, pH, temperature, magnetic fields, and electrical current.

The screening of an aptamer library against the target may be performedby any method known in the art. In one aspect, the target may beimmobilized on a solid support and the aptamers may be added underconditions that allow binding of aptamers with low specificity. Unboundaptamers may be washed from the target with a series of washes ofincreasing stringency. Aptamers that remain bound to the target throughthe wash steps may be sequenced and amplified for further rounds ofselection or used for the design of additional aptamers with highsequence similarity. Several rounds of target binding, washing,sequencing and amplification, or design of new aptamers, may be repeateduntil aptamers of desired specificity and binding affinity aregenerated. An aptamer library may also be screened using a bead-basedapproach utilizing beads which each include multiple copies of anaptamer. An aptamer library may also be screened using an array-basedapproach, for example by spotting multiple copies of each aptamer of thelibrary onto an array and then assessing the spots to which the targetbinds. An aptamer library may also be screened using a particle displayapproach. For example, beads or other particles that are attached toaptamers can be arrayed on a support to form an array. In someembodiments, an aptamer library may be screened using a single moleculeprotein array.

In some cases, the fraction or percentage of the targets to which anidentified detectable probe or affinity reagent binds may be measured,for example by comparing the number of bound copies of the probe withthe number of polypeptides available for binding. In some embodiments, adetectable probe or affinity reagent may bind to at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80% or more of the polypeptides including thetarget. Additionally, once a particular detectable probe or affinityreagent is identified and selected, it may be validated. In someembodiments, a selected detectable probe or affinity reagent may bevalidated against a plurality of sequences containing epitopes to whichthe detectable probe or affinity reagent is characterized as binding. Insome embodiments, a selected detectable probe or affinity reagent may bevalidated by assessing the selected detectable probe or affinity reagentagainst a plurality of protein sequences on a single molecule proteinarray.

The detectable probe or affinity reagent may be attached to detectablelabels before or after the selection and characterization steps. In somecases, the detectable probe or affinity reagent may be attached tolabel(s) before the initial selection step. In some cases, thedetectable probe or affinity reagent may be attached to label(s) afterthe initial selection step and before the characterization steps. Insome cases, the detectable probe or affinity reagent may be attached tolabel(s) after the selection and characterization steps. In some cases,the detectable probe or affinity reagent may be attached to first labelsfor the selection step, and attached to second labels for thecharacterization steps. The second labels may be added with the firstlabels, or instead of the first labels. In some cases, a first label maybe used for the selection and characterization steps, and a second labelused to create a final affinity reagent or detectable probe.

Detection and Observability

A detectable probe of the present disclosure may be configured toprovide a detectable signal that imparts observability to the probe. Theobservability may refer to the property of producing a physical signalthat can be readily sensed by a detection device at a level that exceedsthe background or noise of a detection system. For example, afluorescent probe might be considered observable if it produces afluorescent signal intensity that exceeds a background fluorescentsignal intensity by a threshold amount, e.g., 10%, 50%, 100%, 2×, 3×,4×, 5×, or more. In another example, a nucleic acid barcode signal mightbe considered observable if it produces a threshold number of sequencecounts (e.g., by next-generation sequencing or array-basedhybridization) with or without amplification, e.g., 1000 or moresequence counts after 10 rounds of signal amplification. In general,observability may be influenced by several factors, including: 1)likelihood of a detectable probe binding a target; 2) strength ofbackground signal; and 3) signal-dampening mechanisms. Factors that mayinfluence the likelihood of a detectable probe binding a target caninclude those set forth above.

A background signal may refer to a detectable signal that has the sameor similar characteristics as compared to the desired signal. Forexample, in a fluorescent label system, a background signal may includedetected radiation at the same wavelength or within a radiationwavelength range as the anticipated fluorescent signal from afluorescent label, e.g., a background fluorescent signal may be detectedbetween 485 nm and 495 nm when attempting to observe an Alexa-Fluor® 488dye. For radiative signals, a background signal may arise due to naturalfluorescence, natural luminescence, or autofluorescence of a material,transmission, reflection, or refraction of external radiation at adetected wavelength, or residual signal left by fluorescent probes froma prior detection process.

Background radiative signal may be spatially homogeneous or spatiallyheterogeneous. For some characterization assays, a radiative backgroundsignal may be measured before, during, or after an assay. For example,the spatial composition of a material may be characterized by multiplecycles of applying one or more detectable probes to the material. Oversuccessive cycles, residual detectable probes may be randomly left onthe material, creating a heterogeneous background signal duringsuccessive detection cycles. A residual detectable probe may eventuallydissociate, meaning that the background signal may have spatial andtemporal heterogeneity. In another example, the same above-describedmaterial characterization assay may occur in a detection system that isnot optically sealed, permitting some external radiation to be detectedin the system. The external radiation does not have a uniformdistribution over the material, leading to detection of a non-homogenousgradient or distribution of radiation in the background signal. Theexternal radiation may not vary over time, causing a spatiallyheterogeneous but temporally homogeneous background.

A detectable probe may be characterized as producing a detectable signalthat exceeds a background signal threshold. A luminescently labeled,detectable probe may be characterized as having a fluorescent signal (orother type of luminescent signal) that exceeds a background fluorescentsignal. A luminescent signal may be quantified, for example by totalphoton count over a fixed period of time, to obtain a background signalor a detected probe signal. A quantified luminescent signal may bemeasured at a point, location, or address, or over a region including aplurality of points, locations, or addresses. A detectable probe may beconfigured to have a detectable signal that exceeds a thresholdbackground signal level, such as an average signal over an entire regionor a maximum signal within a region. The overall strength of signal froma detectable probe may be controlled by the total number of signalinglabels (e.g., fluorophores) attached to the detectable probe. Thespatial strength of a probe (i.e., the signal strength at a specificlocation) may be controlled by the density of signaling labels (e.g.,fluorophores) attached to the probe.

A detectable probe may be configured to produce a detectable signal thatexceeds expected or observed background signal. For example, adetectable probe that is configured to bind to a material or to abinding partner on the material may produce a detectable signal thatexceeds the measured background signal produced by the material. Adetectable probe may produce a detectable signal that exceeds abackground signal by a particular amount. Conversely, a detectable probemay produce a detectable signal that is less than a detectable limitdepending upon the mode of signal sensing. A detectable probe mayproduce a detectable signal intensity that exceeds a background signalintensity by at least about 1.5×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×,11×, 12×, 13×, 14×, 15×, 16×, 17×, 18×, 19×, 20×, 25×, 30×, 40×, 50×,75×, 100×, or more. Alternatively or additionally, a detectable probemay have a detectable signal intensity that exceeds a background signalintensity by no more than about 100×, 75×, 50×, 40×, 30×, 25×, 20×, 19×,18×, 17×, 16×, 15×, 14×, 13×, 12×, 11×, 10×, 9×, 8×, 7×, 6×, 5×, 4×, 3×,2×, 1.5×, or less.

For radiative signals (e.g., fluorescence), signal-dampening may arisedue to one or more mechanisms that remove or interfere with theradiative signal. Exemplary signal-dampening mechanisms may includequenching, self-quenching, photo-bleaching, and label loss. Quenchingmay occur due to the presence of chemical species that inhibit theemission of or that absorb emitted photons. Some species in acharacterization system may inherently absorb emitted photons, leadingto a reduction or quenching of radiative signal. In some configurations,particular species may be added to a detection system (e.g., oxygenscavengers such as ascorbate) that inhibit the formation of species thatmay quench a radiative signal. A specific form of quenching isself-quenching, where a luminescent signal from a luminophore may bequenched by a second luminophore of the same or similar species.Self-quenching may be related to inter-label configurations, such as thespacing between adjacent luminophores and the relative orientation ofluminophores. In some configurations, luminescent signal may decrease asthe spacing between adjacent luminophores decreases. In otherconfigurations, luminescent signal may increase as the spacing betweenadjacent luminophores decreases (e.g., Forster resonant energytransfer).

Quenching, self-quenching, and related optical phenomena may becharacterized by an effective quantum yield. Individual fluorophores mayhave a characteristic or measured quantum yield. The effective quantumyield of a detectable probe composition including a plurality offluorophores may be a measured or characterized quantum yield underassay conditions. A detectable probe composition may have an effectivequantum yield of at least about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4,0.45, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60,0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71, 0.72,0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.80, 0.81, 0.82, 0.83, 0.84,0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96,0.97, 0.98, 0.99, or more. Alternatively or additionally, a detectableprobe composition may have an effective quantum yield of no more thanabout 0.99, 0.98, 0.97, 0.96, 0.95, 0.94, 0.93, 0.92, 0.91, 0.90, 0.89,0.88, 0.87, 0.86, 0.85, 0.84, 0.83, 0.82, 0.81, 0.80, 0.79, 0.78, 0.77,0.76, 0.75, 0.74, 0.73, 0.72, 0.71, 0.70, 0.69, 0.68, 0.67, 0.66, 0.65,0.64, 0.63, 0.62, 0.61, 0.60, 0.59, 0.58, 0.57, 0.56, 0.55, 0.54, 0.53,0.52, 0.51, 0.50, 0.45, 0.4, 0.35, 0.3, 0.25, 0.2, 0.15, 0.1, or less.

A radiative signal may also be dampened or otherwise diminished by achemical mechanism such as photo-bleaching or label loss. Withoutwishing to be bound by theory, photo-bleaching may occur due toirreversible chemical changes to a label, such as a fluorescent label.Photo-bleaching may be due to any known mechanism, includinginteractions of photons with a fluorophore, interactions of photons withspecies that may damage a fluorophore (e.g., free radicals such astriplet oxygen), and interactions between chemical species andfluorophores that alter or damage a fluorophore. Photo-bleaching andother forms of signal-dampening may be transient or time-relatedphenomena. For example, photo-bleaching due to irradiation may increasein a proportion with total time or total time of irradiation. Likewise,total amount or concentration of disruptive chemically-modifying speciesmay impact a rate of photo-bleaching. For example, photo-bleachingseverity may increase with increased photon count or photon density. Arate of signal loss due to photo-bleaching or another chemical mechanismmay be constant or variable (e.g., exponential, logarithmic) with time.

A detectable probe composition may experience a variable or increasingloss of signal over time due to quenching, self-quenching,photo-bleaching, label loss, or other mechanisms. A detectable probecomposition may have a rate of signal loss of at least about 0.001%/min,0.01%/min, 0.1%/min, 0.5%/min, 1%/min, 2%/min, 3%/min, 4%/min, 5%/min,6%/min, 7%/min, 8%/min, 9%/min, 10%/min, 15%/min, 20%/min, 25%/min,30%/min, 35%/min, 40%/min, 45%/min, 50%/min, 55%/min, 60%/min, 65%/min,70%/min, 75%/min, 80%/min, 85%/min, 90%/min, 95%/min, or more.Alternatively or additionally, a detectable probe composition may have arate of signal loss of no more than about 95%/min, 90%/min, 85%/min,80%/min, 75%/min, 70%/min, 65%/min, 60%/min, 55%/min, 50%/min, 45%/min,40%/min, 35%/min, 30%/min, 25%/min, 20%/min, 15%/min, 10%/min, 9%/min,8%/min, 7%/min, 6%/min, 5%/min, 4%/min, 3%/min, 2%/min, 1%/min,0.5%/min, 0.1%/min, 0.01%/min, 0.001%/min, or less.

A detectable probe may produce detectable signal that is distinguishablefrom background, for example, by having intensity that exceedsbackground signal or having a detectable characteristic that is resolvedfrom background signal. A detectable probe may be characterized asproducing signal intensity that exceeds background signal for a givenamount of time despite signal degrading mechanisms such asphoto-bleaching and label loss. For example, a fluorescent detectableprobe may have a fluorescent signal that exceeds a backgroundfluorescent signal for at least 10 minutes of continuous ornon-continuous excitation. An observability time may be defined as theminimum length of time that a detectable probe produces a detectablesignal that is distinguishable from a local or average backgroundsignal. In some configurations, observability time may be increased byincreasing the total number of label components on a detectable probe.Observability time may scale proportionally with the number of labelcomponents attached to a detectable probe. The observability time of adetectable probe may be increased or even maximized to ensuredetectability throughout an assay. The observability time of adetectable probe may be limited to within a certain value to enableintended signal removal, such as by photo-bleaching. A detectable probemay be observed for, or have an observability time of, at least about 1s, 15 s, 30 s, 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9min, 10 min, 15 min, 20 min, 30 min, 45 min, 60 min, 90 min, 120 min, ormore. Alternatively or additionally, a detectable probe may be observedfor, or have an observability time of, no more than about 120 min, 90min, 60 min, 45 min, 30 min, 20 min, 15 min, 10 min, 9 min, 8 min, 7min, 6 min, 5 min, 4 min, 3 min, 2 min, 1 min, 30 s, 15 s, 1 s, or less.

A detectable probe may improve the observability of a bindinginteraction by producing a detectable signal that is well in excess of abackground signal. Moreover, a detectable probe may increase theobservability of a binding interaction by improving spatial resolutionof a detectable signal. A detectable probe may be configured to providespatial resolution of a detectable signal in several ways, including: 1)concentration of labels within a small region of a retaining componentto create an optically dense signal source; 2) distribution of labelsover a large region of a retaining component to create an opticallyuniform signal source; or 3) concentration of labels over all or a largefraction of a retaining component to create an optically dense anduniform signal source. FIGS. 12A-12B depict an example of a luminescentsignal (e.g. fluorescent signal) spatial resolution utilizing adetectable probe. FIG. 12A depicts fluorescent count data over a region(e.g., a surface of a material), including background fluorescent count,when a single binding component including a plurality of fluorophores isbound to a binding partner in the center of the region. The region maybe uniformly divided into nine subregions where each subregion may beindividually quantified for fluorescence (e.g., each subregion maycorrespond to a sensor pixel). The number of fluorophores attached tothe affinity reagent may be limited due to the structure and affinity ofthe affinity reagent. The fluorescent counts in FIG. 12A show a modestincrease in the fluorescence count of the center subregion, possibly dueto a binding interaction between the affinity reagent and the bindingpartner, but the signal intensity may not be sufficiently above thebackground counts of the eight surrounding subregions to conclude that abinding interaction occurred. FIG. 12B depicts a similar situation toFIG. 12A, but with a detectable probe including a significantly largernumber of fluorophores than the affinity reagent described for FIG. 12A.The significantly increased fluorescent signal observed in the centralsubregion, along with the increases in signal from the surroundingsubregions (possibly due to signal cross-talk from the centralsubregion), increase the confidence that the observed signal is due to abinding interaction of the detectable probe with the binding partner.

A detectable probe may be sized to provide a detectable signal over aregion of a solid support, surface or field of view. The size of aregion over which a detectable probe provides a signal may be determinedby the size of a feature within the region. For example, a detectableprobe may be sized to provide a signal over an area that is larger thanthe area occupied by a binding partner (e.g., a polypeptide). Adetectable probe may be designed to be larger than a binding partner toreduce a background signal, for example by reducing autofluorescencefrom the binding partner or a material adjacent to the binding partner.Alternatively, a detectable probe may be designed to be smaller than abinding partner to increase the spatial resolution of the detectablesignal from the detectable probe.

In some configurations, a detectable probe composition may be designedto produce a detectable signal over an area of a solid support, surfaceor field of view that is larger than the binding partner to which thedetectable probe may bind or have affinity. Observability of the probeover a particular area may be improved by increasing the distributionand/or concentration of label components over a surface of thedetectable probe. The probe may produce a detectable signal over arelatively large area by including additional detection molecules thatenhance the detectable signal provided by the detectable probe. FIG. 13illustrates a detectable probe composition for creating a detectablesignal over a large area relative to the area occupied by the bindingpartner that it recognizes. A detectable probe 1310 binds to a bindingpartner 1330 (e.g., a polypeptide) that is anchored to a surface orsolid support 1370, optionally by an anchoring group 1340 (e.g., anucleic acid such as a structured nucleic acid particle or a functionalgroup linkage). The area of detectable signal produced by the detectableprobe 1310 is increased by joining the detectable probe 1310 withadditional detection molecules 1320. The detection molecules 1320 mayinclude a retaining component and a plurality of label components (e.g.,fluorophores). The retaining components of the detection molecules 1320need not be attached to binding components. However, the retainingcomponents of the detection molecules 1320 can be attached to bindingcomponents other than those present in detectable probe 1310. Thedetection molecules 1320 are joined to the detectable probe 1310 bylinkers 1325. The linkers 1325 may be permanent linkers (e.g.,heterobifunctional linkers, click reaction products, streptavidin-biotinlinkages) or may be non-permanent linkers (e.g., hybridized nucleicacids). The linkers can be flexible, for example, allowing directinteraction between the detection molecules 1320 and the detectableprobe 1310; or the linkers can be rigid, for example, constraining thedetection molecules 1320 from interacting with the detectable probe1310. The complex formed by the detectable probe 1310 and the detectionmolecules 1320 may be formed prior to the binding of the detectableprobe 1310 to the binding partner 1330, or may be formed by binding ofthe detectable probe, followed by contacting of detection molecules 1320with the detectable probe 1310.

A detectable probe that is configured to bind with binding partner maybe smaller or larger sized relative to the size of the polypeptides.Size can be measured as volume, molecular weight, longest dimension,effective diameter, radius of gyration, hydrodynamic radius, projection(e.g. footprint) or the like. Alternatively, a detectable probe that isconfigured to bind with a binding partner at a site (e.g. a site in anarray of polypeptides) may be smaller or larger sized relative to thesize of the site. A detectable probe or a complex including a detectableprobe may be sized to be at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%,110%, 120%, 130%, 140%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 600%,700%, 800%, 900%, 1000%, or more compared to the size of a bindingpartner or site. Alternatively or additionally, a detectable probe or acomplex including a detectable probe may be sized to be no more thanabout 1000%, 900%, 800%, 700%, 600%, 500%, 400%, 300%, 250%, 200%, 175%,150%, 140%, 130%, 120%, 110%, 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%,60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or lesscompared to the size of the binding partner or site.

The label components of a detectable probe may be configured formultiplex detection by the inclusion of more than one type or species oflabel component on the detectable probe. The ability to attach aplurality of binding components to a detectable probe adds to thedynamic flexibility of the detectable probe for multiplexing. Adetectable probe can have a unique combination of label components thatconstitutes a unique fingerprint or signature for the detectable probe.Binding pools can be created from mixtures of detectable probes withdiffering binding specificities that are indicated by the specific probesignature or fingerprint such that an observed specific bindinginteraction can be determined through the observation of the signatureor fingerprint. A signature or fingerprint may be created by alteringthe number of types or species of binding components as well as theratios of label components attached to the detectable probe. Adetectable probe may include at least about 2, 3, 4, 5, 6, 7, 8, 9, 10,or more different species of label component. Alternatively oradditionally, a detectable probe for a multiplexing composition mayinclude no more than about 10, 9, 8, 7, 6, 5, 4, 3, or fewer differentspecies of label component. The ratio of a first species of labelcomponent to a second species of label component may be at least about1.5:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, or more.Alternatively or additionally, the ratio of a first species of labelcomponent to a second species of label component may be no more thanabout 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1.1:1, or less.

A multiplexed probe composition may include a plurality of detectableprobes with differing binding affinities, where the binding affinity ofany given detectable probe can be determined by the unique fingerprintor signature of label components on the detectable probe. The number ofunique types of detectable probes in a multiplexed probe composition maybe determined by the type of detection system used to observe bindinginteractions and the sensitivity of the detection system to distinguishdifferences in probe signatures or fingerprints. For fluorescent labels,a fluorescent detection system may have a limited number of detectionchannels based upon the fluorescence wavelengths of fluorophores on thedetectable probes. The detection range within each detection channel mayalso constrain the amount of each fluorophore added to a detectableprobe.

A multiplexed probe composition may include at least about 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, or more unique types of detectableprobes. Alternatively or additionally, a multiplexing probe compositionmay include no more than about 50, 49, 48, 47, 46, 45, 44, 43, 42, 41,40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23,22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3,or fewer unique types of detectable probes. Uniqueness may be manifestin the number or diversity of components present in the detectableprobes, such as the number or diversity of binding components present inthe probes, the number or diversity of label components present in theprobes, the number or diversity of retaining components present in theprobes and/or the number or diversity of tags or barcodes present in theprobes.

A detectable probe may be configured to be detected by Forster resonantenergy transfer (FRET). In some configurations, a detectable probe maycontain attached fluorophore pairs that are configured to be detected bya FRET mechanism. Without wishing to be bound by theory, efficient FRETdetection can exploit an absorber fluorophore and an emitter fluorophorethat are positioned within a sufficient distance to allow energy to beefficiently transferred between fluorophores. In some configurations,FRET can provide the advantage of increasing the number of probes thatcan be distinguished from each other using a given excitation source.For example, a first probe can include fluorophore component D thatemits at a first wavelength when excited by the excitation source. Asecond probe can include fluorophore component D and fluorophorecomponent A, in which excitation of fluorophore component D by theexcitation source causes energy transfer to fluorophore component Awhich, in turn, emits at a second wavelength. The two emissionwavelengths can be resolved using standard optics to distinguish the twoprobes from each other.

FIGS. 14A-14B depict various configurations for detectable probes thatcan be detected by a FRET mechanism. FIG. 14A depicts a configurationfor generating FRET fluorophore pairs utilizing the helical structure ofa nucleic acid to position the donor fluorophore and the acceptorfluorophore at a critical pairing distance, Δ_(SFRET). A retainingcomponent may include a continuous nucleic acid strand 1410.Oligonucleotides 1420 may be hybridized to the continuous nucleic acidstrand 1410, creating regions of double-stranded nucleic acids andsingle-stranded nucleic acid 1425. Oligonucleotides may include donorfluorophores 1430 or acceptor fluorophores 1432 in alternating patternsto generate FRET fluorophore pairs at a proper spacing of Δ_(SFRET).FIG. 14B depicts an alternative method of generating FRET fluorophorepairs utilizing a binding molecule that is configured to hybridize to adetectable probe. A detectable probe 1440 may include a plurality ofdonor fluorophores 1430 along a portion of the detectable probe 1440that is configured to bind with a binding molecule 1445. The bindingmolecule 1445 includes a plurality of acceptor fluorophores 1432 thatare aligned along the edge of the binding molecule 1445 that binds withthe detectable probe 1440. The contacting of a detectable probe 1440with a binding molecule 1445 may cause a binding region 1450 to form,aligning the donor fluorophores 1430 and the acceptor fluorophores 1432to form FRET fluorophore pairs that are within the proper FRET spacingof Δ_(SFRET). In some configurations, the binding molecule 1445 may becoupled to a binding partner or a location where a binding partner islocated, thereby permitting the FRET interaction to occur when thedetectable probe 1440 binds to the binding partner and the bindingmolecule 1445 then binds the detectable probe 1440. In someconfigurations the binding molecule 1445 may be a SNAP or othersubstance that is attached to a binding partner for one or more bindingcomponents attached to detectable probe 1440. Accordingly, binding ofthe probe to the binding partner can be determined based on theobservation of FRET between the donor fluorophores 1430 and acceptorfluorophores 1432.

A detectable probe composition may include a coupled pair of donor andacceptor luminophores. Acceptable donor/acceptor pairs may includeCy2/Cy3, Cy3/Cy5, FITC/TRITC, PE/APC, Alexa-Fluor® 488/Alexa-Fluor® 514,Alexa-Fluor® 488/Alexa-Fluor® 532, Alexa-Fluor® 488/Alexa-Fluor® 546,Alexa-Fluor® 488/Alexa-Fluor® 610, Alexa-Fluor® 647/Alexa-Fluor® 680,Alexa-Fluor® 647/Alexa-Fluor® 700, Alexa-Fluor® 647/Alexa-Fluor® 750,Cyan FP/YFP, Cerulean FP/YFP, GFP/YFP, GFP/mRFP, or combinationsthereof.

FRET dye pairs may be coupled to a nucleic acid, such as anoligonucleotide or a scaffold strand that forms a portion of astructured nucleic acid particle. An nucleic acid containing a FRET dyepair may have modified nucleotides to which the dyes are attached spacedsufficiently to allow a FRET interaction to occur. The proper spacingmay be determined by the naturally arising helical structure that ariseswhen an oligonucleotide hybridizes to a scaffold strand. Two adjacentdyes in a FRET pair may be spaced at least about 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleotides apart.Alternatively or additionally, two adjacent dyes in a FRET pair may bespaced no more than about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9,8, 7, 6, 5, 4, 3, or fewer nucleotides apart.

Binding interactions involving detectable probes may be observed usinglabel components other than fluorophores. A detectable probe may includea barcode label component (e.g., a nucleic acid barcode). A barcodelabel component may include a unique sequence (e.g., DNA, RNA, aminoacids) that, when decoded, provides information identifying a detectableprobe species. For example, a plurality of identical detectable probesmay each be labeled with the same species of barcode (i.e. the barcodeshaving the same sequence), thereby providing a uniform and singularsignal of binding interaction for that particular detectable probe. Abarcode signal may be detected and decoded by a method such asnext-generation sequencing to obtain an observation of an interactionbetween a detectable probe and a binding partner, epitope, or targetmoiety.

FIGS. 15A-15D depict a system for utilizing nucleic acid barcodes torecord binding interactions between a detectable probe and a bindingpartner, epitope, or target moiety. FIG. 15A depicts a system containinga detectable probe 1510 including a nucleic acid barcode sequence thatis joined to the detectable probe 1510 by a linker 1520. The nucleicacid barcode sequence includes two priming sequences 1522 and anidentifying sequence 1525 between the two priming sequences 1522. Thedetectable probe 1510 binds a binding partner, epitope, or target moiety1530 that is optionally bound to a solid support 1570. The bindingpartner, epitope, or target moiety 1530 may be bound to the solidsupport 1570 by an anchoring group 1540 (e.g. a SNAP or chemicallinker). The binding partner, epitope, or target moiety 1530 may have anassociated linker 1550 (e.g., attached to the solid support 1570,anchoring group 1540, or binding partner, epitope, or target moiety1530). The associated linker 1550 may be terminated with a complementarypriming sequence 1552 that forms a hybridized bond with a primingsequence 1522 of the detectable probe 1510 nucleic acid barcode. Thecomplex formed by the detectable probe 1510 and the binding partner,epitope, or target moiety 1530 is contacted with polymerase enzyme 1560that is configured to bind a nucleic acid. FIG. 15B shows the binding ofthe polymerase 1560 to the hybridized nucleic acid sequence formed bythe joining of the priming sequence 1522 and the complementary primingsequence 1552 to initiate an extension reaction. FIG. 15C depicts afinal step of the extension reaction. The identifying sequence 1525 andthe priming sequence 1522 have been added by extension onto the terminalsequence of the associated linker 1550 to add a complementaryidentifying sequence 1555 and an additional complementary primingsequence 1552. FIG. 15D depicts the binding partner, epitope, or targetmoiety 1530 after multiple cycles of detectable probe 1510 binding andprimer extension. Each probe adds a unique identifying sequence (e.g.,1555, 1556, 1557) to the sequence at the end of the linker 1550. Theaddition of a complementary priming sequence 1552 at the end of eachidentifying sequence (1555, 1556, 1557) permits subsequent binding ofthe linker 1550 to a detectable probe. Detectable probe 1510 is shownwith an optional plurality of label components (shown as 6 pointedstars). It will be understood that the detectable probe need not includelabels, for example, in configurations where identifying sequences aredecoded to determine the biding history for target moiety 1530.Accordingly, an affinity reagent can be configured or used asexemplified in FIGS. 15A-15D.

Other examples of tags that can be attached to an affinity reagent ofthe present disclosure and methods for using and detecting the tags, forexample, in assays for detecting, sequencing or quantifying polypeptidesare set forth in US Pat App. Pub. Nos. 2020/0348308 A1, 2020/0348307 A1,or 2019/0145982 A1, each of which is incorporated herein by reference.

Methods of Fabricating Detectable Probes and Affinity Reagents

Detectable probes or affinity reagents as described in the presentdisclosure may be fabricated by a suitable method. Fabrication of adetectable probe or affinity reagent may include one or more of thefollowing steps: 1) creating a retaining component that is configured toattach a plurality of binding components and/or a plurality of labelcomponents; 2) attaching one or more binding components to the retainingcomponent; 3) attaching one or more label components to the retainingcomponent; and 4) attaching additional components to the retainingcomponent.

Retaining components may be obtained through a fabrication process.Non-nucleic acid retaining components (e.g., polymers, metal, ceramic,carbon, or semiconductor nanoparticles) may be fabricated through a bulkfabrication and/or purification process. Following the production ofnon-nucleic acid retaining components, one or more processing steps mayoccur to add one or more surface functionalities to the retainingcomponents. The functionalities may be added for the purposes ofimproving retaining component solvent solubility properties or providingattachment sites for binding components and/or label components. Surfacefunctionalities may include functional groups (e.g., functional groupsconfigured to undergo a click reaction) that are configured to attachbinding components and/or label components, or nucleic acids that areconfigured to hybridize with a complementary nucleic acids containing anattached binding component and/or label component. For example,retaining components including silicon or silicon dioxide nanoparticlesmay be functionalized with silanized compounds to covalently add aplurality of functionalities to the silicon-containing surface of theparticle. After functionalization of non-nucleic acid retainingcomponents, affinity groups may be joined to the retaining components byany suitable technique, such as click reactions or nucleic acidhybridization.

Fabrication of nucleic acid retaining components (e.g., nucleic acidorigami, nucleic acid nanoballs) may be formed by conventionaltechniques. Nucleic acid nanoballs may be fabricated by a method such asrolling circle amplification to generate a scaffold strand that may befurther modified to attach a plurality of binding components and/orlabel components. Exemplary methods for making nucleic acid nanoballsare described, for example, in U.S. Pat. No. 8,445,194, which isincorporated herein by reference. Nucleic acid retaining componentsincluding nucleic acid origami may be fabricated, for example, usingtechniques described in Rothemund, Nature 440:297⁻³⁰² (2006) and U.S.Pat. Nos. 8,501,923 and 9,340,416, each of which is incorporated hereinby reference.

FIG. 21A shows a first pathway to forming a detectable probe with anucleic acid retaining component. Oligonucleotides with attached bindingcomponents 2120 and oligonucleotides with attached label components 2130are prepared before the retaining component is assembled. Theoligonucleotides with attached binding components 2120 andoligonucleotides with attached label components 2130 are contacted witha single-stranded scaffold 2110 (e.g., M13 phage DNA, plasmid DNA) andadditional structural nucleic acids 2140. The nucleic acids arecontacted in a suitable DNA buffer at an elevated temperature (e.g., atleast about 50° C., 60° C., 70° C., 80° C., or 90° C.), then cooled.Oligonucleotides will hybridize to the scaffold strand 2110 at theappropriate sequence-dependent positions to form a detectable probe2150.

FIG. 21B shows an alternative pathway to forming a detectable probe witha nucleic acid retaining component. Oligonucleotides with handles thatare configured to attach binding components 2125 and oligonucleotideswith handles that are configured to attach label components 2135 areprepared before the retaining component is assembled. Theoligonucleotides with handles that are configured to attach bindingcomponents 2125 and oligonucleotides with handles that are configured toattach label components 2135 are contacted with a single-strandedscaffold 2110 (e.g., M13 phage DNA, or plasmid DNA) and additionalstructural nucleic acids 2140. The nucleic acids are contacted in asuitable buffer at an elevated temperature (e.g., at least about 50° C.,60° C., 70° C., 80° C., or 90° C.), then cooled. After cooling, aretaining component 2155 that is configured to bind a plurality ofbinding components and/or label components is formed. The retainingcomponent 2155 is contacted with a plurality of binding components 2128and/or label components 2138 that have complementary handles to thehandles on the retaining component 2155 in a suitable attachment buffer.After attachment of the plurality of binding components 2128 and/or theplurality of label components 2138, detectable probe 2150 is formed.

In some configurations, a detectable probe or affinity reagent may beformed by the attachment of a binding component and/or a label componentby the reaction of a functional group configured to form a bond withanother molecule or group, e.g., a bioorthogonal reaction or clickchemistry (see, for example, U.S. Pat. Nos. 6,737,236 and 7,427,678,each incorporated herein by reference in its entirety); azide alkyneHuisgen cycloaddition reactions, which use a copper catalyst (see, forexample, U.S. Pat. Nos. 7,375,234 and 7,763,736, each incorporatedherein by reference in its entirety); Copper-free Huisgen reactions(“metal-free click”) using strained alkynes or triazine-hydrazinemoieties which can link to aldehyde moieties (see, for example, U.S.Pat. No. 7,259,258, which is incorporated by reference); triazinechloride moieties which can link to amine moieties; carboxylic acidmoieties which can link to amine moieties using a coupling reagent, suchas EDC; thiol moieties which can link to thiol moieties; alkene moietieswhich can link to dialkene moieties that are coupled through Diels-Alderreactions; and acetyl bromide moieties which can link to thiophosphatemoieties (see, for example, WO 2005/065814, which is incorporated byreference). A functional group may be configured to react via a clickreaction (e.g., metal-catalyzed azide-alkyne cycloaddition,strain-promoted azide-alkyne cycloaddition, strain-promotedazide-nitrone cycloaddition, strained alkene reactions, thiol-enereaction, Diels-Alder reaction, inverse electron demand Diels-Alderreaction, [3+2] cycloaddition, [4+1] cycloaddition, nucleophilicsubstitution, dihydroxylation, thiol-yne reaction, photoclick, nitronedipole cycloaddition, norbornene cycloaddition, oxanobornadienecycloaddition, tetrazine ligation, tetrazole photoclick reactions).Exemplary silane-derivative CLICK reactants may include alkenes,alkynes, azides, epoxides, amines, thiols, nitrones, isonitriles,isocyanides, aziridines, activated esters, and tetrazines (e.g.,dibenzocyclooctyne-azide, methyltetrazine-transcyclooctylene,epoxide-thiol, etc.). A click reaction may provide an advantageousmethod of rapidly forming a bond under benign conditions (e.g., roomtemperature, aqueous solvents).

In some configurations, a retaining component or other component of adetectable probe or affinity reagent may include different species offunctional groups. The use of different functional groups can provide alevel of control over the number and location of different componentsthat will be attached to the detectable probe or affinity reagent. Inparticular configurations the different functional groups demonstrateorthogonal reactivity, whereby a first component has a moiety that isreactive for a first functional group on the probe but not substantiallyreactive with a second functional group on the probe, and whereby asecond component has a moiety that is reactive for the second functionalgroup but not the first functional group. Accordingly, the number ofdifferent binding components and their locations can be adjusted byappropriate use of orthogonal functional groups on a detectable probe oraffinity reagent, or the number of different label components and theirlocations can be adjusted by appropriate use of orthogonal functionalgroups on a detectable probe or affinity reagent. Moreover, bindingcomponents can be located differently from label components on adetectable probe or affinity reagent by appropriate use of orthogonalfunctional groups, respectively, on the detectable probe or affinityreagent.

Retaining components including non-nucleic acids may be formed byappropriate methods. Of particular interest are methods that permit adegree of spatial control in attaching binding components, labelcomponents, or other components to the retaining component during probeassembly. Spatial control may include the ability to separate orsegregate probe components, or vary the orientation of probe components.For example, spatial control may include separating binding componentsand/or label components from adjacent or neighboring binding componentsand/or label components. In another example, spatial control may includesegregating regions of attached binding components from regions ofattached label components. In yet another example, spatial control mayinclude controlling the relative dispersion or distribution of bindingcomponents and/or labeling components, e.g., providing bindingcomponents and labeling components in a region of a retaining componentat a 10:1 ratio.

FIGS. 32A-32B depict a scheme for controlling probe component locationduring the assembly of a detectable probe or affinity reagent includinga non-nucleic acid retaining component. FIG. 32A depicts a plurality ofparticles 3210 (e.g., nanoparticles, nanobeads, nanospheres, etc.) thatare configured to associate with an interface 3220, such as a multiphaseboundary (e.g., air/liquid interface, oil/water interface). Theinterface 3220 is formed between a first fluid medium 3222 and a secondfluid medium 3224. The portions of each particle of the plurality ofparticles 3210 are exposed to different modification chemistriesdepending upon exposure to the first liquid medium 3222 or second liquidmedium 3224. The portions of each particle of the plurality of particles3210 that are exposed to the first liquid medium 3222 form a firstplurality of functional groups 3235 on the surface of the particles. Theportions of each particle of the plurality of particles 3210 that areexposed to the second liquid medium 3224 form a second plurality offunctional groups 3230 on the surface of the particles. The firstplurality of functional groups 3235 may provide an attachment site for afirst type of probe component (e.g., binding components) and the secondplurality of functional groups 3230 may provide an attachment site for asecond type of probe component (e.g., label components).

FIGS. 32C-32E depict a scheme for controlling probe component locationduring the assembly of a detectable probe or affinity reagent includinga non-nucleic acid retaining component. FIG. 32C depicts a plurality ofparticles 3210 (e.g., nanoparticles, nanobeads, nanospheres, etc.) thatare partially embedded or fixed within a medium 3226. A coating or layer3240 (e.g., a metal, metal oxide, polymer, or hydrogel) is applied tothe exposed portions of each particle of the plurality of particles3210. As shown in FIG. 32D, after a coating or layer 3240 has beenapplied to the plurality of particles 3210, the medium 3226 may beremoved, thereby providing a plurality of particles 3210 with a partialcoating or layer 3240. As shown in FIG. 32E, the differing surfacechemistries of the uncoated and coated portions of the plurality ofparticles 3210 may be used to differentially functionalize the pluralityof particles 3210 with a partial coating or layer 3240. The coating orlayer 3240 may be provided with a first plurality of functional groups3235 that can provide an attachment site for a first type of probecomponent (e.g., binding components). The uncoated portions of eachparticle may be provided with a second plurality of functional groups3230 that can provide an attachment site for a second type of probecomponent (e.g., binding components).

The location or positioning of binding components and/or labelcomponents on a non-nucleic acid retaining component may also becontrolled by controlling the spatial or surface density of attachmentsites on the surface of the non-nucleic acid retaining component. FIG.33 depicts a preparation process for a particle or nanoparticleretaining group. A particle or nanoparticle 3310 is combined with amixture of moieties that are to be attached to the particle ornanoparticle 3310 surface, such as binding component attachment sites3320, label component attachment sites 3330, or modifying groups 3340.The ratios of components within the mixture of moieties is balanced toensure proportional modification of the particle or nanoparticle 3310surface. The final result is a retaining component with homogeneous ornear-homogeneous distribution of each component over the particle ornanoparticle 3310 surface based upon the component concentration in themixture of moieties. Alternatively, components can be added sequentiallyby initially adding a two-component mixture consisting of a first typeof attachment site and blocking groups. After surface functionalization,the blocking groups can be completely or partially removed to providesurface sites for attaching other types of attachment sites.

A plurality of affinity reagents, a plurality of detectable probes, or acombination of at least one affinity reagent and at least one detectableprobe may be conjugated to form a multi-probe complex. Similarly,detectable probes or affinity reagents may be configured with one ormore coupling groups that permit attachment of a reagent or probe to oneor more other reagents or probes. The coupling groups may be configuredto form a covalent interaction, a non-covalent interaction, anelectrostatic interaction, a magnetic interaction, or any otherinteraction that forms an association between detectable probes affinityreagents or both. An association between two or more probes in amulti-probe complex may be weak, temporary, or reversible. Anassociation between two or more probes in a multi-probe complex may bestrong, permanent, or irreversible. In some cases, detectable probes oraffinity reagents may be coupled by hybridization of complementarynucleic acid strands or streptavidin-biotin coupling groups. In othercases, detectable probes or affinity reagents may be covalently coupledby, for example a click reaction or cross-linking (e.g., chemical orphoto-initiated cross-linking).

A multi-probe complex may be formed as a portion of a probe synthesisprocess. Formation of probes or retaining components may facilitate thelocation, orientation, and attachment of probe components, such asbinding components and/or label components. FIG. 37A-37C depicts aprocess for utilizing a complex to tune the location of bindingcomponents on non-nucleic acid retaining components. FIG. 37A depictstwo particle retaining components 3710 (e.g., nanoparticles) that forman association with a phase boundary 3720 formed between a first medium3722 and a second medium 3724. The retaining components 3710 areconfigured to form a complex by an attractive interaction betweenparticles (e.g., an electrostatic or magnetic attraction, misciblesurface functionalities, etc.). The retaining components 3710 arecontacted with a plurality of binding components 3730 within the secondmedium 3724 that are configured to attach to attachment sites on theparticles 3710. FIG. 37B depicts the complexed particles 3710 after theplurality of binding components 3730 have attached to the particlesurface within the second medium 3724. The binding components 3730 havebeen limited to certain regions of the particle 3710 surfaces due toexclusion of surface area within the first medium 3722 and exclusion ofsurface near the inter-particle association region. FIG. 37C shows anoptional process in which the interaction between the particles isbroken, releasing individual detectable probes 3740. In otherconfigurations, the particles may be retained as a multi-probe complex.

Nucleic acid-based retaining components (e.g. a scaffold as set forth inU.S. Provisional Application No. 63/112,607) may be synthesized throughstandard nucleic acid synthesis chemistries, and be provided withspecific label groups, e.g., through incorporation of prelabelednucleotides, or by providing attachment sites for label groups to beadded subsequently, e.g., providing functional side groups, as well asone or more sites for coupling to the binding component. Likewise, a PEGscaffold may be synthesized and functionalized with functional groupswhich will allow the attachment of either, or both of, bindingcomponents or label components. In another example, a polypeptidescaffold may be synthesized including groups which will allow for theattachment of either, or both of, probe components or label components.In some cases, a retaining component may be considerably larger than thelabel to which it is attached. In other cases, the retaining componentmay be of a similar size as the label component to which it is attached.In other cases, the label component may be larger than the retainingcomponent to which it is attached.

In some cases a retaining component, such as a nucleic acid scaffold,may be synthesized directly upon a binding component. For example, areagent used for synthesis of the retaining component can include thebinding component. In some cases a retaining component, such as anucleic acid scaffold, may be synthesized directly upon a linkermolecule that is to be attached to the binding component. For example, areagent used for synthesis of the retaining component can include thelinker. In some embodiments, a retaining component may be produced by atemplate mediated polymerase extension reaction using a nucleotide mixin which some or all of the nucleotides that are to be incorporated havea label or a functional moiety for attaching a label component. Forexample, three nucleotide species used by the polymerase duringextension can be unlabeled (and/or can lack a functional group), and thefourth nucleotide species can be labeled with a fluorescent moiety orother label moiety (or the fourth nucleotide can have the functionalgroup). In this example, the complementary base of the fourth nucleotidecan occur in a predetermined pattern in the template. For example, thecomplementary base of the fourth nucleotide may occur in at a nucleotidespacing set forth elsewhere herein.

As shown in FIG. 49A, a binding component, illustrated here as a nucleicacid aptamer, may include a primer sequence at one terminus, e.g., the3′ terminus. A template nucleic acid, e.g., as described above, thatincludes a sequence segment complementary to the primer sequence maythen be hybridized to the primer segment of the binding component (seeFIG. 49B, Step 1). Polymerase mediated template based extension of theprimer sequence on the binding component in the presence of theunlabeled and labeled nucleotides, e.g. as described above, then resultsin an affinity probe that includes the binding component (e.g., theaptamer), along with the label component that includes the nucleic acidof the retaining component with the incorporated labels (the extensionproduct of the polymerase reaction with labeled nucleotides (See FIG.49B, Step 2). In some cases, the template may include a specificnucleotide species, e.g., a guanosine nucleotide at desired intervals orin desired locations, while the extension reaction is carried out in thepresence of unlabeled adenosine, thymidine and guanosine triphosphates,and labeled cytosine triphosphate. This results in periodicincorporation of the labeled cytosine nucleotides into the extensionproduct scaffold. For example, by including a template with a guanosinenucleotide at every 3^(rd), 4^(th) 5^(th), etc. position in thetemplate, one can allow for incorporation of a labeled cytosine at every3^(rd), 4^(th), 5^(th), etc. position of the retaining component. Aswill be appreciated, the specific labeled nucleotide or the periodicityof the incorporated label may be selected depending upon particularneeds of the analysis. In some cases, following this synthesis, thetemplate nucleic acid may remain in place to provide a double strandedlabel portion of the affinity probe, for example, as set forth elsewhereherein.

In some cases, the template for the scaffold may include a circularnucleic acid, where extension of a primer sequence produces an extendedconcatemer having duplicated sequence segments, e.g., through rollingcircle amplification. In an optional format, a template sequence may beprovided coupled to the binding component. A primer may be hybridized tothe template and extended by polymerization in the presence of labeledand unlabeled nucleotides, in order to create a second, associatedstrand that includes label groups as described above.

In some cases, affinity reagents or detectable probes may be constructedusing a modular format that allows more targeted tailoring of theend-product labeled probe. For example, in some cases, bindingcomponents, e.g., aptamers or antibodies, may be produced and maintainedas unlabeled libraries. In such cases, these binding components may bemaintained with attachment regions for coupling label components to thebinding components in accordance with a desired labeling protocol for agiven experiment. Such attachment regions may include chemical couplingmoieties, e.g., NHS esters, “click” chemistry components (see, e.g., H.C. Kolb; M. G. Finn; K. B. Sharpless (2001), “Click Chemistry: DiverseChemical Function from a Few Good Reactions” Angewandte ChemieInternational Edition. 40 (11): 2004-2021), and other routine chemicalcoupling approaches, where the labeling component includes any necessarycomplementary coupling moieties. An advantage of this modularity is thata variety of different detectable probes or affinity reagents can bereadily formed using the same or similar retaining component.Accordingly, a plurality of different detectable probes or affinityreagents can differ with regard to one or more of the number labelcomponents, variety of label components, number of binding components,and variety of binding components, while having retaining componentsthat share a common structural characteristic. The common structuralcharacteristic can be, for example, size of the retaining component,shape of the retaining component, chemical composition of the retainingcomponent, nucleic acid sequence of the retaining component,three-dimensional structure of an origami in the retaining component,three-dimensional structure of a scaffold in an origami in the retainingcomponent or the like. In some configurations, a plurality of differentdetectable probes can differ with regard to the number of stapleoligonucleotides annealed in an origami structure, the location whereone or more staple oligonucleotides are annealed to a scaffold strand inan origami structure, the length of one or more staple oligonucleotidesannealed to a scaffold strand in an origami structure, the sequence ofone or more staple oligonucleotides annealed to a scaffold strand in anorigami structure, or the number, type or location of functional groupsin an origami structure.

In some cases, binding components may include one member of a binding orcoupling pair (e.g. a receptor), while the label components include acomplementary member of the binding or coupling pair (e.g. a ligand forthe receptor). For example, in some cases, a binding component may becoupled to a single strand nucleic acid sequence while the labelcomponent is coupled to a second strand having a sequence that iscomplementary to the single strand. In such cases, coupling can becarried out through hybridization of the label bound nucleic acid strandto the binding component bound nucleic acid strand. As will beappreciated, in the case of aptamer binding components, production ofthe aptamer and the label coupling component may be produced using,e.g., PCR amplification of the single stranded probe and label couplingcomponent, followed by removal of the complementary strand prior tohybridization of the label component.

In any of the above contexts, label components and retaining componentsmay be synthesized separately from the binding component, either withlabeling groups attached, or as a functionalized retaining component towhich label components may be subsequently attached either before orafter coupling to the binding component. In the case of nucleicacid-based retaining components, such structures may generally besynthesized through well known solid phase nucleic acid synthesistechniques, where known nucleotides are added in succession to buildpolynucleotide structures, or through polymerase chain reactionamplification of a scaffold sequence. As noted, such processes mayemploy periodic introduction of labeled nucleotides during synthesis inorder to build a multi-labeled probe in the form desired. Alternatively,modified nucleotides may be incorporated during synthesis that alloweasy addition of label components post synthesis.

Covalent attachment of a separately synthesized retaining component toanother component (e.g. binding component, label component or anotherretaining component) may be accomplished via chemical or biochemicalapproaches, e.g., through chemical coupling of a nucleic acid scaffoldto the binding component, e.g., using known chemical coupling techniquessuch as click chemistry, or through biochemical methods such as ligationof a nucleic acid scaffold to a nucleic acid component of a bindingcomponent. Alternatively, a retaining component may be non-covalentlyattached to the binding component through hybridization to acomplementary nucleic acid component that is already coupled to thebinding component. An example of such attachment is schematicallyillustrated in FIG. 51 . As shown, the binding component 400 includes anucleic acid probe sequence 400 b attached to it. A separatelysynthesized labeled nucleic acid retaining component 401 having asequence complementary to the probe sequence 400 b is then hybridized tothe binding component to provide a labeled affinity probe.

Any of a variety of covalent or non-covalent chemistries can be used toattach or join components of a detectable probe or affinity reagent setforth herein. Chemistries and methods set forth herein in the context ofattaching retaining components to other components can also be used toattach detectable probes or affinity reagents to other substances suchas binding partners (e.g. polypeptides), surfaces, solid supports, sitesof an array, or particles. Moreover, the chemistries and methods can beused to synthesize retaining components, binding components or labelcomponents, or to add functional groups or linkers to such components.

In some configurations that employ polypeptide-based binding components,e.g., antibodies or antibody fragments, a coupling approach may beemployed for coupling of only a single label component to a singlebinding component through incorporation of a single coupling group to agiven polypeptide. In particular, a binding component, such as anantibody or antibody fragment may be provided with a first couplinghandle or functional group, while a separate label component (orcomponent that may be readily labeled) is provided with a secondcoupling handle or functional group that reacts with or binds to thefirst coupling moiety to achieve attachment between the bindingcomponent and the label component.

One example of such an approach is a SpyTag/SpyCatcher approach tolabeling (See, e.g., Zakeri B, Fierer J O, Celik E, Chittock E C,Schwarz-Linek U, Moy V T, Howarth M (March 2012). “Peptide tag forming arapid covalent bond to a protein, through engineering a bacterialadhesin”. Proceedings of the National Academy of Sciences of the UnitedStates of America. 109 (12): E690-7). In this approach, a 13 amino acidtag polypeptide (SpyCatcher) forms a first coupling handle, with a 12.3kDa protein (Spy Tag) forming the other coupling handle. By way ofexample, the Spy Catcher may be integrated into a first component (e.g.a binding component or label component) as a recombinant fusion protein.The Spy Catcher component irreversibly binds to the Spy Tag through anisopeptide bond, which may be fluorescently or otherwise labeled fordetection. As will be appreciated either the Tag or the Catcher may beintegrated with the first component.

In some cases, different components may be attached to each other usingclick chemistry. In cases of components having nucleic acid moietiesthese may be attached using ligation and or hybridization. In somecases, a linker is used for attachment. Examples of linkers includedouble stranded DNA, single stranded DNA, or different molecular weightpolyethylene glycol. Linkers may also include functional groups thatallow for bioconjugation.

In some cases, attachment can employ chemical conjugation,bioconjugation, enzymatic conjugation, photo-conjugation,thermal-conjugation, or a combination thereof. (Spicer, C. D., Pashuck,E. T., & Stevens, M. M., Achieving Controlled Biomolecule-BiomaterialConjugation. Chemical Reviews., 2018, 118, Pgs. 7702-7743, and Greg T.Hermanson, “Bioconjugate Techniques”, Academic Press; 3^(rd) Edition,2013, herein incorporated by reference for this disclosure). Forexample, bioconjugation may be used to form a covalent bond between twomolecules, at least one of which is a biomolecule. In some cases, twocomponents of a detectable probe or affinity reagent that are to beattached to each other may be functionalized. Functionalizing bothpartners may improve the efficiency or speed of an attachment (e.g.conjugation) reaction. For example, a sulfhydryl group (—SH) or amine(—NH₂) of a chemically active site of an aptamer, biological, orchemical entity may be functionalized to allow for greater reactivity orefficiency of an attachment reaction. Any of a variety ofsulfhydryl-reactive (or thiol-reactive) or amine conjugation chemistriesmay be used to couple chemical moieties to sulfhydryl or amine groups.Examples include, but are not limited to, use of haloacetyls,maleimides, aziridines, acryloyls, arylating agents, vinylsulfones,pyridyl disulfides, TNB-thiols and/or othersulfhydryl-reactive/amine-reactive/thiol-reactive agents. Many of thesegroups attachto sulfhydryl groups through either alkylation (e.g., byformation of a thioether or amine bond) or disulfide exchange (e.g., byformation of a disulfide bond). More strategies and detail regardingreactions for bioconjugation are described down below and may beextended to other appropriate molecules.

Attachment can be accomplished in part by a chemical reaction of achemical moiety or linker molecule with a chemically active site on abiomolecule or other substance. The chemical conjugation may proceed viaan amide formation reaction, reductive amination reaction, N-terminalmodification, thiol Michael addition reaction, disulfide formationreaction, copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC)reaction, strain-promoted alkyne-azide cycloaddtion reaction (SPAAC),Strain-promoted alkyne-nitrone cycloaddition (SPANC), inverseelectron-demand Diels-Alder (IEDDA) reaction, oxime/hydrazone formationreaction, free-radical polymerization reaction, or a combinationthereof. Enzyme-mediated conjugation may proceed via transglutaminases,peroxidases, sortase, SpyTag-SpyCatcher, or a combination thereof. Photoconjugation and activation may proceed via photoacrylate cross-linkingreaction, photo thiol-ene reaction, photo thiol-yne reaction, or acombination thereof. In some cases, attachment or conjugation mayproceed via noncovalent interactions, these may be throughself-assembling peptides, binding sequences, host-guest chemistry,nucleic acids, or a combination thereof.

In some cases, site-selectivity methods may be employed to modifyreaction moieties of detectable probes, affinity reagents or componentsthereof to increase attachment efficiency, ease of use, and/orreproducibility. Three common strategies can be employed forsite-selective attachment. (i) Modification strategies that can select asingle motif among many, rather than targeting a generic functionalgroup or moiety. This may be determined by surrounding sequence, localenvironment, or subtle differences in reactivity. The ability of enzymesto modify a specific amino acid within a protein sequence or a glycan ata single position are particularly prominent. Reactions that displayexquisite chemo-selectivity also fall within this category, such asthose that target the unique reactivity of the protein N-terminus or theanomeric position of glycans. (ii) The site-specific incorporation ofunnatural functionalities, by hijacking native biosynthetic pathways maybe utilized. (iii) The installation of unique reactivity via chemicalsynthesis may be utilized. The complete or partial synthesis ofpolypeptides and oligonucleotides is widespread, particularly usingsolid-phase approaches. These techniques allow access to sequences of upto 100 amino acids or 200 nucleotides, with the ability to install awide variety of functionalized monomers with precise positional control.

In some cases, chemical conjugation techniques may be applied forcreating attached species such as biomaterial-biomolecule conjugates.Functional groups used for attachment may be native to a substance thatis to be modified (e.g. a biomolecule that is to be modified) or may beincorporated synthetically. In the illustrations below, R and R′ may bea biomolecule (for example, but not limited to: SNAP, proteins, nucleicacids such as nucleic acid origami or nucleic acid nanoballs,carbohydrates, lipids, metabolites, small molecules, monomers,oligomers, polymers), affinity reagent, detectable probe, bindingcomponent, label component, retaining component, and/or a solid support.

In some cases, reductive amination may be utilized for attachment suchas attachment via bioconjugation. Amines can react reversibly withaldehydes to form a transient imine moiety, with accompanyingelimination of water. This reaction takes place in rapid equilibrium,with the unconjugated starting materials being strongly favored inaqueous conditions due to the high concentration of water. However, in asecond step the unstable imine can be irreversibly reduced to thecorresponding amine via treatment with sodium cyanoborohydride. Thismild reducing reagent enables the selective reduction of imines even inthe presence of unreacted aldehydes. As a result, irreversibleconjugation of a biomolecule or other substance can gradually occur to asecond substance such as a biomaterial of interest. In contrast,stronger reducing agents such as sodium borohydride are also able toreduce aldehydes. This two-step reductive amination process can also beutilized for the modification of ketones. For example, reductiveamination has therefore been primarily used for the modification ofsodium periodate-treated alginate and chitosan scaffolds. The order ofreactivity may also be reversed for the attachment of reducing sugars,by exploiting the terminal aldehyde/ketone generated in the open-chainform. This strategy, for example, may be exploited to mimic theglucosylation and galactosylation patterns of native collagen in ECM,via reductive amination of maltose and lactose respectively.

In some cases, isothiocyanates may be used to attach substances to eachother. For example, isothiocyanates of a biomolecule or solid supportmay be utilized for bioconjugation. An isothiocyanate moiety may reactwith nucleophiles such as amines, sulfhydryls, the phenolate ion oftyrosine side chains or other molecules to form a stable bond betweentwo molecules.

In some cases, an isocyanate may be utilized for attachment of twosubstances. For example, an isocyanate of a biomolecule or solid supportmay be utilized for bioconjugation. For example, isocyanates can reactwith amine-containing molecules to form stable isourea linkages.

In some cases, an acyl azide may be utilized for attachment of twosubstances. For example, an acyl azide of a biomolecule or solid supportmay be utilized for bioconjugation. For example, acyl azide areactivated carboxylate groups that can react with primary amines to formamide bonds.

In some cases, an amide may be utilized for attachment of twosubstances. For example, an amide of a biomolecule or solid support maybe utilized for bioconjugation. For example, the use of reactiveN-hydroxysuccinimide (NHS) esters is particularly widespread. WhileNHS-esters can be preformed, often they are instead generated in situthrough the use of N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide(EDC) coupling chemistry and coupled directly to the species ofinterest. Although formation of the activated NHS-ester is favored undermildly acidic conditions (pH ˜5), subsequent amide coupling isaccelerated at higher pH wherein the amine coupling partner is notprotonated. One-step modification at an intermediate pH of ˜6.5 ispossible. Attachment is typically undertaken by first forming the activeNETS-ester at pH 5, before raising the pH to ˜8 and adding the aminecoupling partner in a two-step procedure. In some cases, water-solublederivative sulfo-NHS may be utilized as an alternative. In some cases,NHS esters of a biomolecule or other substance can react and couple withtyrosine, serine, and threonine —OH groups as opposed to N-terminal αamines and lysine side-chain ε-amines.

In some cases, a sulfonyl chloride may be utilized for attachment of twosubstances. For example, a sulfonyl chloride of a biomolecule or solidsupport may be utilized for bioconjugation. For example, reaction of asulfonyl chloride compound with a primary amine-containing moleculeproceeds with loss of the chlorine atom and formation of a sulfonamidelinkage.

In some cases, a tosylate ester may be utilized for attachment of twosubstances. For example, a tosylate ester of a biomolecule or solidsupport may be utilized for bioconjugation. For example, functionalgroups including tosylate esters can be formed from the reaction of4-toluenesulfonyl chloride (also called tosyl chloride or TsCl) with ahydroxyl group to yield the sulfonyl ester derivative. The sulfonylester may couple with nucleophiles to produce a covalent bond and mayresult in a secondary amine linkage with primary amines, a thioetherlinkage with sulf-hydryl groups, or an ether bond with hydroxyls.

In some cases, a carbonyl may be utilized for attachment of twosubstances. For example, a carbonyl of a biomolecule or solid supportmay be utilized for bioconjugation. For example, carbonyl groups such asaldehydes, ketones, and glyoxals can react with amines to form Schiffbase intermediates which are in equilibrium with their free forms. Insome cases, the addition of sodium borohydride or sodiumcyanoborohydride to a reaction medium containing an aldehyde compoundand an amine-containing molecule will result in reduction of the Schiffbase intermediate and covalent bond formation, creating a secondaryamine linkage between the two molecules.

In some cases, an epoxide or oxirane may be utilized for attachment oftwo substances. For example, an epoxide or oxirane of a biomolecule orsolid support may be utilized for bioconjugation. For example, anepoxide or oxirane group may react with nucleo-philes in a ring-openingprocess. The reaction can take place with primary amines, sulfhydryls,or hydroxyl groups to create secondary amine, thioether, or ether bonds,respectively.

In some cases, a carbonate may be utilized for attachment of twosubstances. For example, a carbonate of a biomolecule or solid supportmay be utilized for bioconjugation. For example, carbonates may reactwith nucleophiles to form carbamate linkages, disuccinimidyl carbonate,can be used to activate hydroxyl-containing molecules to formamine-reactive succinimidyl carbonate intermediates. In some cases, thiscarbonate activation procedure can be used in coupling polyethyleneglycol (PEG) to proteins and other amine-containing molecules. In somecases, nucleophiles, such as the primary amino groups of proteins, canreact with the succinimidyl carbonate functional groups to give stablecarbamate (aliphatic urethane) bonds

In some cases, an aryl halide may be utilized for attachment of twosubstances. For example, an aryl halide of a biomolecule or solidsupport may be utilized for bioconjugation. For example, aryl halidecompounds such as fluorobenzene derivatives can be used to form covalentbonds with amine-containing molecules like proteins. Other nucleophilessuch as thiol, imidazolyl, and phenolate groups can also react to formstable bonds. In some cases, fluorobenzene-type compounds have been usedas functional groups in homobifunctional crosslinking agents. Forexample, their reaction with amines involves nucleophilic displacementof the fluorine atom with the amine derivative, creating a substitutedaryl amine bond.

In some cases, an imidoester may be utilized for attachment of twosubstances. For example, an imidoester of a biomolecule or solid supportmay be utilized for bioconjugation. For example, the α-amines andε-amines of proteins may be targeted and crosslinked by reacting withhomobifunctional imidoesters. In some cases, after conjugating twoproteins with a bifunctional imidoester crosslinker, excess imidoesterfunctional groups may be blocked with ethanolamine.

In some cases, a carbodiimide may be utilized for attachment of twosubstances. For example, carbodiimides may be utilized forbioconjugation. Generally, carbodiimides are zero-length crosslinkingagents that may be used to mediate the formation of an amide orphosphoramidate linkage between a carboxylate group and an amine or aphosphate and an amine, respectively. Carbodiimides are zero-lengthreagents because in forming these bonds no additional chemical structureis introduced between the conjugating molecules. In some cases,N-substituted carbodiimides can react with carboxylic acids to formhighly reactive, O-acylisourea derivatives. This active species may thenreact with a nucleophile such as a primary amine to form an amide bond.In some cases, sulfhydryl groups may attack the active species and formthioester linkages. In some cases, hydrazide-containing compounds canalso be coupled to carboxylate groups using a carbodiimide-mediatedreaction. Using bifunctional hydrazide reagents, carboxylates may bemodified to possess terminal hydra-zide groups able to conjugate withother carbonyl compounds.

In some cases, a phosphate may be utilized for attachment of twosubstances. For example, a biomolecule containing phosphate groups, suchas the 5′ phosphate of oligonucleotides or a phosphorylated amino acidof a polypeptide, may also be conjugated to amine-containing moleculesby using a carbodiimide-mediated reaction. For example, the carbodiimideof a biomolecule may activate the phosphate to an intermediate phosphateester similar to its reaction with carboxylates. In the presence of anamine, the ester reacts to form a stable phosphoramidate bond.

In some cases, an acid anhydride may be utilized for attachment of twosubstances. For example, an acid anhydride of a biomolecule or solidsupport may be utilized for bioconjugation. Anhydrides are highlyreactive toward nucleophiles and are able to acylate a number of theimportant functional groups of proteins and other molecules. Forexample, protein functional groups able to react with anhydrides includebut not limited to the α-amines at the N-terminals, the E-amine oflysine side chains, cysteine sulfhydryl groups, the phenolate ion oftyrosine residues, and the imid-azolyl ring of histidines. In somecases, the site of reactivity for anhydrides in protein molecules ismodification of any attached carbohydrate chains. In some cases, inaddition to amino group modification in a polypeptide chain,glycoproteins may be modified at their polysaccharide hydroxyl groups toform esterified derivatives.

In some cases, a fluorophenyl ester may be utilized for attachment oftwo substances. For example, a fluorophenyl ester of a biomolecule orsolid support may be utilized for bioconjugation. Flurophenyl esters canbe another type of carboxylic acid derivative that may react with aminesconsists of the ester of a fluorophenol compound, which creates a groupcapable of forming amide bonds with proteins and other molecules. Insome cases, fluorophenyl esters may be: a pentafluorophenyl (PFP) ester,a tetrafluorophenyl (TFP) ester, or a sulfo-tetrafluoro-phenyl (STP)ester. In some cases, fluorophenyl esters react with amine-containingmolecules at slightly alkaline pH values to give the same amide bondlinkages as NHS esters.

In some cases, a hydroxymethyl phosphine may be utilized for attachmentof two substances. For example, a hydroxymethyl phosphine of abiomolecule or solid support may be utilized for bioconjugation.Phosphine derivatives with hydroxymethyl group substitutions may act asattachment agents for coupling or crosslinking purposes. For example,tris(hydroxymethyl) phosphine (THP) andβ-[tris(hydroxymethyl)phos-phino] propionic acid (THPP) are smalltrifunctional compounds that spontaneously react with nucleophiles, suchas amines, to form covalent linkages.

In some cases, a thiol may be utilized for attachment of two substances.For example, the thiol reactivity of a biomolecule or solid support maybe utilized for bioconjugation. For example, the thiol group of cysteineis the most nucleophilic functional group found among the 20proteinogenic amino acids. Through careful control of pH, selectivemodification over other nucleophilic amino acid residues such as lysinecan be readily achieved. Another example, thiol modification ofoligonucleotides may be used to enable derivatization, though the easewith which alternative functional groups with enhanced chemicalorthogonality can be installed has limited use forbiomaterial-conjugation. Further, the conjugate addition of thiols toα,β-unsaturated carbonyls, also known as Michael addition, may be usedto form polypeptide conjugates in the fields of tissue engineering,functional materials, and protein modification. In general, reactionrates and conjugation efficiencies are primarily controlled by threefactors and may be modified as needed: (i) the pK_(a) of the thiol; (ii)the electrophilicity of the Michael-acceptor; (iii) the choice ofcatalyst. Regarding (i): the thiolate anion is the active nucleophileduring Michael addition, and the propensity of the thiol to undergodeprotonation may determine thiolate concentration and thus reactionrates. For example, the lower pK_(a) of aromatic thiols, when comparedto their aliphatic counterparts, leads to a higher rate of reaction ratea weak base is used to catalyze the. As a result, local structure cansignificantly alter conjugation efficiency, particularly for polypeptidesubstrates. The pK_(a) and reactivity of cysteine containing peptidescan be altered significantly through rational choice of surroundingamino acids, the presence of positively charged amino acids, such aslysine and arginine, acts to lower the thiol pK_(a) and thus enhancereactivity. Regarding (ii): the Michael-acceptor becomes more electrondeficient it becomes more activated toward nucleophilic attack, and thusreaction rates increase. Within the most widely utilized acceptors inthe biomaterial field, a trend of reactivity can be generalized asmaleimides>vinyl sulfones>acrylates>acrylamides>methacrylates. Regarding(iii) Michael additions can be accelerated by either basic ornucleophilic catalysis (although both act by increasing theconcentration of the active thiolate).

In some cases, the unique nucleophilicity of thiols can be exploited forselective reaction with a number of alternative electrophiles, whichallow efficient and selective attachment to be achieved. For example,one such group includes α-halocarbonyls, with iodoacetamide basedreagents finding particular utility. Higher thiol selectivity may beachieved using less electrophilic bromo- and even chloro-derivatives,though reactivity is also drastically reduced. More recently,methylsulfonyl heteroaromatic derivatives have emerged as promisingreagents for thiol-specific conjugation. In other cases, alternativethiol-functional groups, such as disulfide-bridging pyridazinediones,carbonylacrylic reagents, and cyclopropenyl ketones may be utilized forbioconjugation.

In some cases, a sulfhydryl may be utilized for attachment of twosubstances. For example, sulfhydryl of a biomolecule or solid supportmay be utilized for bioconjugation. In some cases, three forms ofactivated halogen derivatives can be used to create sulfhydryl-reactivecompounds: haloacetyl, benzyl halides, and alkyl halides. In each ofthese compounds, the halogen group may be easily displaced by anattacking nucleophilic substance to form an alkylated derivative withloss of HX (where X is the halogen and the hydrogen comes from thenucleophile). Haloacetyl compounds and benzyl halides typically areiodine or bromine derivatives, whereas the halo-mustards mainly employchlorine and bromine forms. Iodoacetyl groups have also been usedsuccessfully to couple affinity ligands to chromatography supports.

In some cases, a maleimide may be utilized for attachment of twosubstances. For example, a maleimide of a biomolecule or solid supportmay be utilized for bioconjugation. The double bond of maleimides mayundergo an alkylation reaction with sulfhydryl groups to form stablethioether bonds.

In some cases, an aziridine may be utilized for attachment of twosubstances. For example, an aziridine of a biomolecule or solid supportmay be utilized for bioconjugation. The highly hindered nature of thisheterocyclic ring gives it strong reactivity toward nucleophiles. Forexample, sulfhydryls will react with aziridine-containing reagents in aring-opening process, forming thioether bonds. The simplest aziridinecompound, ethylenimine, can be used to transform available sulfhydrylgroups into amines. In some cases, substituted aziridines may be used toform homobifunctional and trifunctional crosslinking agents.

In some cases, thiol-maleimide reactions are particularly useful forundertaking conjugation at low concentrations or when requiringextremely high efficiencies due to the value of the biomoleculesubstrate. The use of maleimides for attachment is further enhanced bythe ease with which they may be introduced into a wide range ofmaterials, through the modification of amines with the difunctionalreagent succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate,commonly referred to by its abbreviation SMCC. For example, this reagenthas been widely used to first introduce a maleimide functional group ona biomaterial of choice and then to enable the attachment of bothpeptides and growth factors to produce bioactive scaffolds.

In some cases, an acryloyl may be utilized for attachment of twosubstances. For example, an acryloyl of a biomolecule or solid supportmay be utilized for bioconjugation. The reactive double bonds arecapable of undergoing additional reactions with sulfhydryl groups. Insome cases, the reaction of an acryloyl compound with a sulfhydryl groupoccurs with the creation of a stable thioether bond. In some cases, theacryloyl has found use in the design of the sulfhydryl-reactivefluorescent probe, 6-acryloyl-2-dimethylaminonaphthalene.

In some cases, an aryl group may be utilized for attachment of twosubstances. For example, an aryl group of a biomolecule or solid supportmay be utilized for bioconjugation with a sulfhydryl group. Althougharyl halides are commonly used to modify amine-containing molecules toform aryl amine derivatives, they also may react quite readily withsulfhydryl groups. For example, fluorobenzene-type compounds have beenused as functional groups in homobifunctional crosslinking agents. Theirreaction with nucleophiles involves bimolecular nucleophilicsubstitution, causing the replacement of the fluorine atom with thesulfhydryl derivative and creating a substituted aryl bond. Conjugatesformed with sulfhydryl groups are reversible by cleaving with an excessof thiol (such as DTT).

In some cases, a disulfide may be utilized for attachment of twosubstances. For example, the disulfide group of a biomolecule or solidsupport may be utilized for bioconjugation. In some cases, compoundscontaining a disulfide group are able to participate in disulfideexchange reactions with another thiol. The disulfide exchange (alsocalled interchange) process involves attack of the thiol at thedisulfide, breaking the —S—S— bond, with subsequent formation of a newmixed disulfide including a portion of the original disulfide compound.The reduction of disulfide groups to sulfhydryls in proteins usingthiol-containing reductants proceeds through the intermediate formationof a mixed disulfide. In some cases, crosslinking or modificationreactions may use disulfide exchange processes to form disulfidelinkages with sulfhydryl-containing molecules.

In some cases, disulfide bonds may be utilized for attachment such asbioconjugation. For example, the use of disulfide exchange reactions maybe favored for modifying polypeptides of interest. The most commonlyused reagents in tissue engineering are based upon reactivepyridylthio-disulfides, which undergo rapid thiol-exchange to releasethe poorly nucleophilic and spectroscopically active 2-mercaptopyridine.Additionally, due to the reversible nature of disulfide bond formation,cleavage can be controlled with temporal precision by the addition ofreducing agents such as dithiothreitol (DTT) or glutathione.

In some cases, an pyridyl dithiol may be utilized for attachment of twosubstances. For example, a pyridyl dithiol functional group may be usedin the construction of crosslinkers or modification reagents forbioconjugation. Pyridyl disulfides may be created from available primaryamines on molecules through the reaction of 2-iminothiolane in tandemwith 4,4′-dipyridyl disulfide. For instance, the simultaneous reactionamong a protein or other molecule, 2-iminothiolane, and 4,4′-dipyri-dyldisulfide yields a modification containing reactive pyridyl disulfidegroups in a single step. A pyridyl disulfide will readily undergo aninterchange reaction with a free sulfhydryl to yield a single mixeddisulfide product.

In some cases, sulfhydryl groups activated with the leaving group5-thio-2-nitrobenzoic acid can be used to couple free thiols bydisulfide interchange similar to pyridyl disulfides, as describedherein. The disulfide of Ellman's reagent readily undergoes disulfideexchange with a free sulfhydryl to form a mixed disulfide withconcomitant release of one molecule of the chromogenic substance5-sulfido-2-nitroben-zoate, also called 5-thio-2-nitrobenzoic acid(TNB). The TNB-thiol group can again undergo interchange with asulfhydryl-containing target molecule to yield a disulfide crosslink.Upon coupling with a sulfhydryl compound, the TNB group is released.

In some cases, disulfide reduction may be performed usingthiol-containing compounds such as TCEP, DTT, 2-mercaptoethanol, or2-mercaptoethylamine.

In some cases, an vinyl sulfone may be utilized for attachment of twosubstances. For example, a vinyl sulfone group of a biomolecule or solidsupport may be utilized for bioconjugation. For example, the Michaeladdition of thiols to activated vinyl sulfones to formbiomolecule-material conjugates have been used to demonstrate thatcysteine capped peptides could cross-link vinyl-sulfone functionalizedmultiarm PEGs to form protease responsive hydrogels, enabling cellinvasion during tissue growth. In some cases, in addition to thiols,vinyl sulfone groups can react with amines and hydroxyls under higher pHconditions. The product of the reaction of a thiol with a vinyl sulfonegives a single stereoisomer structure. In addition, crosslinkers andmodification reagents containing a vinyl sulfone can be used to activatesurfaces or molecules to contain thiol-reactive groups.

In some cases, thiol-containing molecules can interact with metal ionsand metal surfaces to form dative bonds for bioconjugation. In somecases, oxygen- and nitrogen-containing organic or biomolecules may beused to chelate metal ions, such as in various lanthanide chelates,bifunctional metal chelating compounds, and FeBABE. In addition, aminoacid side chains and prosthetic groups in proteins frequently formbioinorganic motifs by coordinating a metal ion as part of an activecenter.

In some cases, thiol organic compounds may be used routinely to coatmetallic surfaces or particles to form biocompatible layers or createfunctional groups for further conjugation of substances such asbiomolecules. For instance, thiol-containing aliphatic/PEG linkers havebeen used to form self-assembled monolayers (SAMs) on planar goldsurfaces and particles.

In some cases, a number of alternative coupling systems may be used forattachment between substances or biomolecule functionalization. Theseinclude the use of O-nitrophenyl esters (which possess reduced stabilityin aqueous conditions) or 1,1′-carbonyldiimidazole (CDI) to formamine-bridging carbamate linkages rather than amides. Hydrazines canalso be used in place of amines during EDC/NHS mediated couplings.Hydrazine-functionalized peptides can be coupled to biomaterials in asingle step at pH 5-6. In doing so, a degree of site-selectivity can beachieved over lysine residues present.

In some cases, N-terminal modification of a biomolecule may be utilizedfor bioconjugation. For example, 2-pyridinecarboxaldehyde modifiedacrylamide hydrogels may react specifically with the N-terminus of ECMproteins, forming a cyclic imidazolidinone product with the adjacentamide bond and enabling the orientated display of these keybioinstructive motifs.

In some cases, acrylates, acrylamides, and methacrylates of a substancesuch as a biomolecule or solid support may be utilized for attachment.In some cases, thiolynes of a substance such as a biomolecule or solidsupport may be utilized for bioconjugation.

In some cases, thiol-reactive conjugation such as native chemicalligation (NCL) can be utilized to attach substances via peptide bondformation, for example, to attach peptides and proteins to biomaterialscaffolds. For example, a peptide having a C-terminal thioester reactswith an N-terminal cysteine residue in another peptide to undergo atrans-thioesterification reaction, which results in the formation of anintermediate thioester with the cysteine thiol.

In some cases, strong binding of (strept)avidin to the small moleculebiotin may be used for attachment. In some cases, (strept)avidin may beattached to a first substance and biotin may be attached to a secondsubstance such that the substances can become attached via binding ofthe (strept)avidin to the biotin. In some cases, modification reagentscan add a functional biotin group to proteins, nucleic acids, and othermolecules. In some cases, depending on the functionality present on thebiotinylation compound, specific functional groups on antibodies orother proteins may be modified to create a (strept)avidin binding site.Amines, carboxylates, sulfhydryls, and carbohydrate groups can bespecifically targeted for biotinylation through the appropriate choiceof biotin derivative. In some cases, photoreactive biotinylationreagents are used to add nonselectively a biotin group to moleculescontaining no convenient functional groups for modification. In somecases, biotin-binding proteins can be immobilized onto surfaces,chromatography supports, microparticles, and nanoparticles for use incoupling biotinylated molecules. In some cases, a series of(strept)avidin-biotin interactions can be built upon each other toutilize the multivalent nature of each tetrameric (strept)avidinmolecule and enhance the detection capability for the target. In somecases, amine-reactive biotinylation reagents that may contain functionalgroups off biotin's valeric acid side chain are able to form covalentbonds with primary amines in proteins and other molecules. In somecases, NHS esters spontaneously react with amines to form amide linkageswhereas carboxylate-containing biotin compounds can be coupled to aminesvia a carbodiimide-mediated reaction using EDC. In some cases,NHS-iminobiotin can be used to label amine-containing molecules with animinobiotin tag, providing reversible binding potential with avidin orstreptavidin. In some cases, Sulfo-NHS-SS-biotin (also known asNHS-SS-biotin) issulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate, along-chain cleavable bio-tinylation reagent that can be used to modifyamine-containing proteins and other molecules. In some cases,1-biotinamido-4-[4′-(maleimidomethyl) cyclohexane-carboxamido]butane, abiotinylation reagent containing a maleimide group at the end of anextended spacer arm reacts with sulfhydryl groups in proteins and othermolecules to form stable thioether linkages. In some cases,N-[6-(biotinamido)hexyl]-3′-(2′-pyridyldithio)propionamide where thereagent contains a 1,6-diaminohexane spacer group which is attached tobiotin's valeric acid side chain, the terminal amino group of the spacermay be further modified via an amide linkage with the acid precursor ofSPDP to create a terminal, sulfhydryl-reactive group. The pyridyldisulfide end of biotin-HPDP may react with free thiol groups inproteins and other molecules to form a disulfide bond with loss ofpyridine-2-thione.

In some cases, a carboxylate may be utilized for attachment of twosubstances. For example, a carboxylate of a biomolecule or solid supportmay be utilized for bioconjugation. In some cases, diazomethane andother diazoalkyl derivatives may be used to label carboxylate groups. Insome cases, N,N′-Carbonyl diimidazole (CDI) may be used to react withcarboxylic acids under nonaqueous conditions to form N-acylimidazoles ofhigh reactivity. An active carboxylate can then react with amines toform amide bonds or with hydroxyl groups to form ester linkages. Inaddition, activation of a styrene/4-vinylbenzoic acid copolymer with CDImay be used to immobilize an enzyme lysozyme or other molecule throughits available amino groups to the carboxyl groups on to a matrix.

In some cases, carbodiimides function as zero-length crosslinking agentscapable of activating a carboxylate group for coupling with anamine-containing compound for attachment. In some cases, carbodiimidesare used to mediate the formation of amide or phosphoramidate linkagesbetween a carboxylate and an amine or a phosphate and an amine.

In some cases, N,N′-disuccinimidyl carbonate or N-hydroxysuccinimidylchloroformate may be utilized to attach species, for example, viabioconjugation. N,N′-Disuccinimidyl carbonate (DSC) consists of acarbonyl group containing, in essence, two NHS esters. The compound ishighly reactive toward nucleophiles. In aqueous solutions, DSC willhydrolyze to form two molecules of N-hydroxysuccinimide (NHS) withrelease of one molecule of CO₂. In nonaqueous environments, the reagentcan be used to activate a hydroxyl group to a succinimidyl carbonatederivative. DSC-activated hydroxylic compounds can be used to conjugatewith amine-containing molecules to form stable crosslinked products.

In some cases, sodium periodate can be used to oxidize hydroxyl groupson adjacent carbon atoms, forming reactive aldehyde moieties suitablefor coupling with amine- or hydrazide-containing molecules forattachment, for example, via bioconjugation. For example, thesereactions can be used to generate crosslinking sites in carbohydrates orglycoproteins for subsequent conjugation of amine-containing moleculesby reductive amination.

In some cases, enzymes may be used to oxidize hydroxyl-containingcarbohydrates to create aldehyde groups for bioconjugation. For example,the reaction of galactose oxidase on terminal galactose orN-acetyl-d-galactose moieties proceeds to form C-6 aldehyde groups onpolysaccharide chains. These groups can then be used for conjugationreactions with amine- or hydrazide-containing molecules.

In some cases, reactive alkyl halogen compounds can be used tospecifically modify hydroxyl groups in carbohydrates, polymers, andother substances for attachment.

In some cases, an aldehyde or ketone may be utilized for attachment oftwo substances. For example, an aldehyde or ketone of a biomolecule orsolid support may be used for bioconjugation. For example, derivativesof hydrazine, especially the hydrazide compounds formed from carboxylategroups, can react specifically with aldehyde or ketone functional groupsin target molecules. To further stabilize the bond between a hydrazideand an aldehyde, the hydrazone may be reacted with sodiumcyanoborohydride to reduce the double bond and form a secure covalentlinkage.

In some cases, an aminooxy may be utilized for attachment of twosubstances. For example, an aminooxy group of a biomolecule or solidsupport may be used for bioconjugation. For example, the chemoselectiveligation reaction that occurs between an aldehyde group and an aminooxygroup yields an oxime linkage (aldoxime) that has been used in manybioconjugation reactions, as well as in the coupling of ligands toinsoluble supports including surfaces. This reaction is also quiteefficient with ketones to form an oxime called a ketoxime.

In some cases, cycloaddition reactions may be utilized for attachment,such as attachment via bioconjugation. In cycloaddition reactions, twoor more unsaturated molecules are brought together to form a cyclicproduct with a reduction in the degree of unsaturation, these reactionpartners are typically absent from natural systems, and so the use ofcycloadditions for conjugation utilizes the introduction of unnaturalfunctionality within a coupling partner.

In some cases, Copper-Catalyzed Azide-Alkyne Cycloadditions (CuAAC) maybe utilized for attachment of two substances. For example, CuAAC may beutilized for bioconjugation. In some cases, the (3+2) cycloadditionbetween an azide and alkyne proceeds spontaneously at high temperatures(>90° C.), producing a mixture of two triazole isomers. In some cases,this reaction proceeds at room temperature, ambient, oxygenated, and/oraqueous environments. In some cases, for example, the formation ofpeptide-material conjugates by CuAAC, using alkyne-capped peptides toform hydrogels with azide-functionalized PEG. In some cases, to achieveconjugation via CuAAC, the copper(I) catalyst can either be addeddirectly, or generated in situ by reduction of an initial copper(II)complex, most commonly using ascorbic acid. The addition of a reducingagent further reduces the sensitivity of the CuAAC ligation to oxygen.Although no additional ligand is necessary for triazole formation, theaddition of tertiary amine based ligands may be used.

In some cases, Strain-Promoted Azide-Alkyne Cycloadditions (SPAAC) maybe utilized for attachment of two substances. SPAAC may be utilized forbioconjugation. In some cases, highly strained cyclooctynes reactreadily with azides to form triazoles under physiological conditions,without the need for any added catalyst. In some cases, in addition tothe use of SPAAC for peptide conjugation, a number of prominent reportshave used SPAAC to attach protein substrates to cyclooctynefunctionalized biomaterials via the introduction of an unnatural azidemotif into the protein coupling partner. In some cases, for example,this is achieved by including maleimide functionalization of nativecysteines present in bone morphogenetic protein-2 (BMP-2), viaenzyme-mediated N-terminal modification of IFN-γ, or via codonreassignment with the unnatural amino acid 4-azidophenylalanine in anumber of protein substrates. In some cases, supramolecular host-guestinteractions can also be used to promote azide-alkyne cycloaddition. Forexample, by bringing two reactive partners into close proximity withinthe cavity of a cucurbit[6]uril host, efficient cycloaddition could beachieved on the surface of proteins, this strategy may be extended toother appropriate molecules.

In some cases, inverse-electron demand Diels-Alder reactions (IEDDA) maybe utilized for attachment, such as attachment via bioconjugation. Forexample, the IEDDA reaction between 1,2,4,5-tetrazines and strainedalkenes or alkynes may be employed. A wide range of suitable derivativesfor undertaking molecule conjugation have been reported, for example, aseries of increasingly strained (and thus reactive) trans-cyclooctenesmay be utilized. In some cases, functionalized norbornene derivativesmay be utilized for undertaking IEDDA reactions. In some cases,triazines may be utilized. In some cases, spirohexene may be utilized.These strategies may be extended to other appropriate molecules. In somecases, hetero-Diels-Alder cycloaddition of maleimides and furans may beutilized for attachment. For example, the coupling offuran-functionalized RGDS peptides (SEQ ID NO: 6) tomaleimide-functionalized PEG-hydrogels may be utilized, this strategymay be extended to other appropriate molecules. In some cases,furan-functionalized hyraluronic acid hydrogels can be cross-linked witha dimaleimide-functionalized peptide via Diels-Alder cycloaddition.

In some cases, oxime and hydrazone may be utilized for attachment of twosubstances. For example, oxime and hydrazone formation may be utilizedfor bioconjugation. In some cases, the stable attachment of peptides andDNA to biomaterials via hydrazone formation can be achieved viadifunctional cross-linking, this strategy may be extended to otherappropriate molecules. For example, protein cross-linked hydrogels canbe produced through oxime modification at both the protein N- andC-termini.

In some cases, the Diels-Alder reaction consists of the covalentcoupling of a diene with an alkene to form a six-membered ring complexfor attachment.

In some cases, transition metal complexes may be utilized forattachment, such as attachment via bioconjugation. The nature of latetransition metals may make a transition metal complex well suited to themanipulation of unsaturated and polarizable functional groups (olefins,alkynes, aryl iodides, arylboronic acids, etc.). For example,Pd(0)-functionalized solid supports may mediate allyl carbamatedeprotections and Suzuki-Miyaura cross-coupling in the cytoplasm. Inother examples, a ruthenium catalyst may be used to mediate allylcarbamate deprotection of a caged fluorophore inside living cells. Insome cases, applications of palladium-based applications in cell cultureinclude copper-free Sonagashira coupling, extracellular Suzuki couplingon the surface of E. coli cells, and conjugation of thiol groups withallyl selenosulfate salts. In some cases, olefin metathesis may beutilized for bioconjugation. For example, with ruthenium complexes,S-allylcysteine can be easily introduced into proteins by a variety ofmethods, including conjugate addition of allyl thiol to dehydroalanine,direct allylation of cysteine, desulfurization of allyl disulfide, ormetabolic incorporation as a methionine surrogate in methionineauxotrophic E. coli.

In some cases, complex formation with boronic acid derivatives may beused for attachment of substances, for example, via bioconjugation. Forexample, boronic acid derivatives are able to form ring structures withother molecules having neighboring functional groups consisting of 1,2-or 1,3-diols, 1,2- or 1,3-hydroxy acids, 1,2- or 1,3-hydroxylamines,1-2- or 1,3-hydroxyamides, 1,2- or 1,3-hydroxyoximes, as well as varioussugars or biomolecules containing these species.

In some cases, enzyme-mediated conjugation may be utilized to attachsubstances. For example, the transglutaminase enzyme family catalyzesthe formation of isopeptide bonds between the primary amine of lysineside chains and the amide bonds of a complementary glutamine residue,this strategy may be extended to other appropriate molecules. In othercases, peroxidase-mediated conjugation may be utilized for conjugation.For example, horse radish peroxidase (HRP) may be utilized to oxidize awide range of organic substrates such as phenol group of tyrosine togenerate a highly reactive radical or quinone intermediate thatundergoes spontaneous dimerization, resulting in the formation of anortho carbon-carbon bond between two tyrosine residues, this strategymay be extended to other appropriate molecules. In some cases shortpeptide tags may be utilized for bioconjugation. These peptide tags maybe as short as 5 amino acids long and may be appended to a polypeptidewhich allows for their subsequent modification.

In some cases, polymerization of low molecular weight monomers may beutilized for attachment of substances, for example, via bioconjugation.Polymerization may be classified as proceeding via one of twomechanisms, either chain-growth or step-growth. During chain-growthpolymerization, monomers are added at the “active” end of a growingpolymer chain, resulting in the formation of high molecular weightmaterials even at low conversions. During step-growth polymerizationsshort oligomer chains couple to form polymeric species, requiring highconversions in order to reach high molecular weights. Both techniquescan be used to form conjugates such as biomolecule-polymer conjugates.The polymerization of acrylate and methacrylate monomers has provenparticularly fruitful. For example, acrylate and methacrylate modifiedproteins can be readily polymerized. Similarly, availability of thesynthetic oligonucleotide phosphoramidite building block “Acrydite”,free-radical polymerization remains one of the most common methodsthrough which to form DNA and RNA functionalized substances. Byundertaking polymerization in the presence of a comonomer, the densityof molecule presentation can be easily tuned, allowing potentialdifficulties from steric hindrance to be overcome. Initiation ofpolymerization can be triggered by a number of means, including heat, UVand visible light, redox reactions, and electrochemistry. Acrylatemodified proteins can also undergo polymerization to produce functionalmaterials, while retaining biological activity. In some cases livingradical polymerizations (LRPs) may be utilized for bioconjugation. Forexample, the most commonly used LRPs for the formation of bioconjugatesinclude atom-transfer radical polymerization (ATRP), reversibleaddition-fragmentation chain transfer (RAFT) polymerization, andnitroxide-mediated polymerization (NMP).

In some cases, photoconjugation may be utilized for attachment ofsubstances, for example, via bioconjugation. In some cases,polymerization is initiated by the production of a radical species,which then propagates through bond formation to create an active polymerchain. The initiation step can be induced via a number of stimuli, withthermal decomposition, redox activation, and electrochemical ionizationof an initiating species being among the most common. Alternatively,many initiators can be activated via light-induced photolytic bondbreakage (type I) or photoactivated abstraction of protons from aco-initiator (type II). Photoinitiation offers the benefits of beingapplicable across a wide temperature range, using narrow and tunableactivation wavelengths dependent on the initiator used, rapidlygenerating radicals, and the ability to control polymerization byremoving the light source. Importantly, the tolerance of polymerizationsto oxygen is greatly enhanced, enabling polymerization in the presenceof cells and tissues. The incorporation of acrylate-functionalizedpeptides and proteins during photopolymerization may be used as a methodfor producing biomaterial conjugates. Alternatively, the photoinitiatedattachment of polypeptides to pendant vinyl groups on preformedmaterials has also been widely reported and more recently used for 3Dpatterning via two-photon excitation. A wide range of photoinitators maybe used in photoconjugation conjugations. For example, but not limitedto, Eosin Y, 2,2-dimethoxy-2-phenyl-acetophenone, Igracure D2959,lithium phenyl-2,4,6-trimethylbenzoylphosphinate, and riboflavin may beused as photoinitiators. Photoinitiators generally absorb light toinitiate the photoreaction processes. In some cases, photoconjugationmay utilize a photo thiol-ene reaction. Thiols can also react withalkenes via a free-radical mechanism. A thiol radical first reacts withan alkene to generate a carbon-centered radical, which can then abstracta proton from another thiol and thus propagate the reaction. Photothiol-ene reactions may be accelerated by electron-rich alkenes, whichgenerate unstable carbon-radical intermediates able to rapidly abstractthiol-hydrogens. Exceptions to this rule are norbornene derivatives, inwhich reactivity is driven instead by the release of ring strain uponthiol addition. This leads to a general trend in reactivity ofnorbornene>vinyl ether>propenyl>allyl ether>acrylate>maleimide.Norbornenes and allyloxycarbonyls (alloc groups) have been particularlywidely used for peptide/protein-biomaterial functionalization, due tothe almost negligible contribution of chain transfer and their ease ofintroduction during peptide synthesis, respectively. For example, analloc group, typically used as an orthogonal lysine protecting groupduring solid-phase peptide synthesis, is an efficient photo thiol-enefunctional group. In other examples, norbornene photo thiol-enereactions may be used for the tethering and spatial patterning ofbioactive peptides and growth factor proteins. In addition to the mostcommonly used alloc and norbornene functional groups, other alkenes havealso been used for biomaterial functionalization. For example, codonreassignment has been used to site-specifically incorporateallyl-cysteine residues into proteins, which can subsequently undergoconjugation through the use of photo thiol-ene reactions. Alternatively,acrylates can undergo mixed-mode photopolymerizations in the presence ofcysteine capped peptides, while allyl disulfide structures have recentlybeen shown to undergo reversible and controlled exchange of conjugatedthiols.

In some cases, aryl azide or halogenate aryl azides can be used toattach substances.

In some cases, a photoreactive group such as benzophenone may beutilized for attachment of substances.

In some cases, photoreactive group anthraquinone may be utilized forattachment of substances, such as attachment via bioconjugation. In somecases, photo thiol-yne reactions may be utilized for attachment ofsubstances, such as attachment via bioconjugation. Most examples ofphoto thiol-yne reactions have exploited simple propargyl-ether or-amine functional groups.

In some cases, photocaging and activation of reactive functionalitiesmay be utilized for attachment of substances, such as attachment viabioconjugation. Generally, a transient reactive species is formedwhether it be an acrylate or thiol derived radical. In some cases,photocaging may be used to mask or protect a functional group until itis desirable for it to be exposed. In some cases, the most widelyutilized cages are based around o-nitrobenzyl and coumarin chromophores.For example, nitrobenzyl-capped cysteine residues may be decaged byirradiation with 325 nm UV light, the released thiol may then react withmaleimide-functionalized peptides via Michael addition, to generate apatterned hydrogel able to guide cell migration. In some cases,6-bromo-hydroxycoumarins may be used for thiol-caging. In some cases,photoaffinitiy probes may be utilized for bioconjugation where a highlyreactive intermediate upon irradiation, which then reacts rapidly withthe nearest accessible functional group with high spatial precision. Insome cases, the most commonly used are phenylazides, benzophenones, andphenyl-diazirines. In some cases, photocaged cycloadditions may be used.For example, the UV irradiation of tetrazoles has been shown to generatea reactive nitrile-imine intermediate which can undergo rapidcycloaddition with electron-deficient alkenes such as acrylates oracrylamides. In some cases, the nitrile-imine side-reactivity withthiols may be utilized for site-specifically conjugation of cysteinecontaining proteins to tetrazole functionalized surfaces.

In some cases, noncovalent interactions may be utilized for attachmentof substances, such as attachment via bioconjugation. In some cases,noncovalent sequences which display a binding affinity for thebiomolecule of interest, allow for postfabrication modification or fornative biomolecules to be simply sequestered from the surroundingswithin biological samples. Useful binding sequences are short peptidesbetween 7 and 20 amino acids in length, derived from a variety ofsources, including known protein binding domains present in vivo ordetermined through techniques such as phage display. In some cases,aptamers can also be used to bind a variety of protein substrates,including the cytokines vascular endothelial growth factor (VEGF) andplatelet derived growth factor (PDGF), as well as cell surface proteinssuch as epidermal growth factor receptor (EGFR). In some cases, bindingsequences can also be introduced into a biomaterial with affinity fornative biopolymers, such as heparin. In some cases, by first inducingbiopolymer binding, the adsorption of an added or endogenous growthfactor or signaling protein to a biomaterial scaffold can then becontrolled. In some cases, binding affinity at the amino acid level canalso be exploited to enable peptide and protein conjugation to certainbiomaterial substrates. For example, the binding of unnaturalcatechol-based amino acids can be used to induce binding to metal oxidecontaining bioglasses and metallic implants, enabling the bioactivity ofthese important technologies to be enhanced.

In some cases, self-assembling peptides may be utilized for attachmentof substances, such as attachment via bioconjugation. For example,native peptides and proteins adopt a series of secondary structures,including β-sheets and α-helices, which can both stabilize individualsequences and control interprotein aggregation. In some cases,self-assembling peptides have been used extensively to assemblehydrogels and fibrous materials. In many of these structures, biologicalepitopes or functional groups can be appended to some or all of thepeptide building blocks during peptide synthesis, to add the desiredbioactivity into the system. Peptide-ligands ranging from simpleadhesion motifs, to laminin derived epitopes, and growth factor mimeticshave all been displayed on the surface of self-assembled fibrils.Alternatively, glycopeptides can be assembled in order to recruitextracellular signaling proteins and growth factors, mimic glycosylationpatterns within hyaluronic acid, or investigate optimal sulfonationratios in glycosaminoglycan scaffolds. In some cases, self-assemblingdomains can also be added to full-length proteins, leading to theincorporation of pendant functionality during hydrogel formation. Insome cases, the propensity of peptides to form secondary structures hasalso been exploited within nonself-assembling scaffolds. This may beachieved by mixing a self-assembling peptide into a covalent hydrogel,composed of either a noninteracting polymer such as interpenetratingnetworks of PEG or systems where additional charge interactions furtherstabilize the final construct, for example between positively chargedpeptides and negatively charged alginate gels. As an alternative,pendant helical groups can be attached to a covalent material and usedto drive the noncovalent attachment of bioactive groups such as growthfactors via self-assembly into coiled-coil triple helices.

In some cases, host-guest chemistry may be utilized for attachment ofsubstances, such as attachment via bioconjugation. For example, theadhesive properties of a β-cyclodextrin modified alginate scaffold couldbe controlled in situ through the addition of a guestnaphthyl-functionalized RGDS peptide (SEQ ID NO: 6) and by subsequentlyintroducing a non-cell adhesive adamantane-RGES peptide (SEQ ID NO: 7)with a higher host binding constant, dynamic modulation of fibroblastcell attachment was enabled. Host-guest interactions betweencyclodextrin and naphthyl- or adamantine-functionalized peptides allowalginate functionalization, this may be applied to other appropriatemolecules.

In some cases, nucleic acids may be utilized for attachment ofsubstances, such as attachment via bioconjugation. In some cases, in ananalogous fashion to self-assembling peptides, nucleic acids can alsoform assembled materials themselves, to generate tunable platforms forthe display of molecules. In some cases, DNA-tagged polypeptides can beconjugated to a suitably functionalized substance.

Generally, incorporating functional groups may be utilized forattachment of substances. For example, introducing uniquely reactivemotifs into molecules provides a chemical “tag” which allows single-siteselectivity or specificity to be achieved. In some cases, polypeptidesor nucleic acids can be produced via solid phase synthesis (SPS). Theversatility of organic synthesis allows difficulties in functional groupincorporation to be overcome, with a wide range of suitablyfunctionalized amino acids and nucleotides available as describedherein. In some cases, an alternative approach is to introduce unnaturalamino acids (UAAs) bearing the desired functional groups. This may beachieved via the modification of lysine residues with amine-reactivederivatives. In some cases, the use of auxotrophic bacterial strains,which are unable to biosynthesize a particular amino acid and thusrequire uptake from the growth media. By starving the bacteria of thenative amino acid and supplementing it with a structurally relatedunnatural analogue, the bacterial cells can be induced to incorporatethe UAA during translation. This technique may be used to install azide-and alkyne-based mimics of methionine, leading to the introduction offunctional groups for undertaking CuAAC and SPAAC reactions. In somecases, the use of codon reassignment using orthogonal tRNA and tRNAsynthetase pairs that selectively recognize and charge an UAA duringtranslation. In some cases, this may be achieved by reassigning theamber stop-codon, UAG, by incorporating a tRNA_(CUA)/tRNA synthetasepair from an alternative kingdom into the host cell. This pair may beable to install the desired UAA, while being effectively invisible tothe endogenous cell machinery. As a result, site-directed mutagenesiscan be used to introduce a single TAG codon at the desired position ofthe coding DNA, leading to the singular introduction of the UAA withhigh specificity and selectivity.

Compositions and Kits

Compositions or kits may be formed including detectable probes oraffinity reagents as described in the present disclosure. Severalconfigurations are set forth below in the context of compositions. Itwill be understood that kits can be similarly configured. A compositionmay include one or more detectable probes or affinity reagents alongwith a solution or solvent, and other constitutive components. Thespecific formulation of a detectable probe or affinity reagentcomposition may depend upon the intended use of the detectable probe oraffinity reagent and the specific composition of a detectable probe oraffinity reagent in the composition. Compositions may be formulatedbased upon several factors, including: 1) stability and storagerequirements for a detectable probe or affinity reagent; 2) propertiesand/or characteristics of the detectable probe or affinity reagent; and3) mode of use for the detectable probe or affinity reagent. Stabilityand/or storage considerations may include conditions to maintainretaining component, binding component, and or label componentstability. Property or characteristic considerations may includeaffinity and/or avidity characteristics, probe dissociation, and labelcomponent characteristics. Mode of use considerations may includedetection methods, multiplexing, and secondary binding interactions.

In general, a composition may include one or more detectable probes oraffinity reagents in a liquid medium. In some configurations, the liquidmedium may be aqueous or otherwise include water. In someconfigurations, the liquid medium may include a non-aqueous solvent,such as a polar solvent or a non-polar solvent. A liquid medium mayinclude a pH buffered solution. A pH buffered solution may be formulatedto maintain solution pH within a desired range, for example to maintainstability of a retaining component or a binding component, or to mediatethe strength of a binding component binding interaction. A liquid mediummay further include one or more salts. Salts in a liquid medium may beadded to alter the ionic strength of the liquid medium. Ionic strengthmay be adjusted to, for example, maintain stability of a retainingcomponent or a binding component, or to mediate the strength of abinding component binding interaction. A probe composition may includean emulsion or colloidal suspension, e.g., a water-in-oil emulsion or anoil-in-water emulsion.

Detectable probes or affinity reagents including nucleic acids (e.g.,DNA origami, DNA nanoballs, aptamers) may be provided in compositionsthat are specifically formulated to maintain nucleic acid stability. Insome configurations, detectable probes or affinity reagents that includenucleic acids may be provided in a liquid medium including a magnesiumsalt (e.g., MgCl₂). The magnesium salt may be provided at a sufficientconcentration to maintain nucleic acid stability (e.g., base-pairbinding, helical structures, etc.). The magnesium salt may have aconcentration of at least about 10 mM, 50 mM, 100 mM, 120 mM, 140 mM,160 mM, 180 mM, 200 mM, 220 mM, 240 mM, 260 mM, 280 mM, 300 mM, 350 mM,400 mM, 500 mM, or more. Alternatively or additionally, the magnesiumsalt may have a concentration of no more than about 500 mM, 400 mM, 350mM, 300 mM, 280 mM, 260 mM, 240 mM, 220 mM, 200 mM, 180 mM, 160 mM, 140mM, 120 mM, 100 mM, 50 mM, 10 mM, or less.

A liquid medium including a detectable probe or affinity reagent mayfurther include a scavenger species. Scavengers may include any chemicalspecies that is intended to remove a chemically detrimental or damagingspecies, such as free-radical scavengers, oxygen scavengers, andmetal-chelating agents. Possible scavengers in a detectable probe oraffinity reagent composition may include species such as hydrazine,ascorbic acid, tocopherol, naringenin, glutathione, stannenes, and EDTA.Scavengers may be included in liquid media that are intended for storageof detectable probe or affinity reagent compositions prior to an assayor other mode of use.

In some configurations, a detectable probe or affinity reagentcomposition that includes a liquid medium may be formulated as amultiphase liquid, such as a water-in-oil emulsion or an oil-in-wateremulsion. A multiphase liquid medium may permit localization and/orconfinement of detectable probes or affinity reagents before or during amode of use. For example, the release of detectable probes or affinityreagents may be controlled by the breaking of an emulsion, therebyreleasing probes confined within the emulsion. Additionally, multiphaseformulations may increase the stability of detectable probes or affinityreagents with mixed chemical characteristics (e.g., hydrophobic andhydrophilic components) or sensitivity to aqueous chemistry (e.g.,susceptibility to hydrolysis).

In some configurations, a detectable probe or affinity reagentcomposition may be formulated to include a competitor species. Acompetitor species may include an affinity reagent or other moleculethat is configured to bind to a binding partner, epitope, or targetmoiety. A competitor species may be characterized by low affinity for abinding partner, epitope, or target moiety. In some configurations, acompetitor species may be characterized by a low affinity for a bindingpartner, epitope or target moiety that is identical to a bindingpartner, epitope, or target moiety for a detectable probe or affinityreagent. In other configurations, a competitor species may becharacterized by a low affinity for a plurality of binding partners,epitopes, or target moieties (e.g., reduced binding specificity).Without wishing to be bound by theory, a competitor species may includea binding species whose displacement from a binding partner, epitope, ortarget moiety is driven by an increase in the Gibbs free energy ofbinding. For example, a competitor species may have a larger bindingenthalpy for a binding partner than a detectable probe or affinityreagent, so it is energetically favorable to displace the competitor andbind the probe. Likewise, a competitor species may increase the entropyof a system by dissociating from a binding partner in favor of adetectable probe or affinity reagent. A competitor species may beadvantageous for creating tunable avidity in detectable probe oraffinity reagent compositions because: 1) a competitor species maythermodynamically encourage the binding of a detectable probe oraffinity reagent to a binding partner, epitope, or target moiety, and 2)a competitor species may affect the kinetics of probe binding byoffering concentration-dependent competition for a binding partner,epitope, or target moiety. FIGS. 20A-20B depict the use of detectableprobe or affinity reagent competition including a binding competitor toencourage binding. FIG. 20A shows the contacting of a binding partner2010 including an epitope or target moiety 2020 with a detectable probe2040. The surface of the binding partner 2010 may be bound with aplurality of competitor affinity reagents 2030 that bindnon-specifically to the surface. FIG. 20B shows free competitor affinityreagents 2035 that are displaced by the binding of the detectable probe2040 to the epitope or target moiety 2020. The displacement of thecompetitor affinity reagents may energetically or entropically encouragethe binding of the detectable probe to the binding partner 2010.

A competitor species may include an affinity reagent such as an aptamer,peptamer, designed ankyrin repeat protein (DARPin), antibody, orantibody fragment. A competitor species may be provided as a componentof a detectable probe or affinity reagent or as a species that isseparate from any detectable probe or affinity reagent. FIG. 16A-16Billustrate detectable probe compositions including a competitor species.FIG. 16A illustrates a detectable probe composition including acompetitor species in free solution. The composition includes adetectable probe 1610 that contains a plurality of attached bindingcomponents 1620 (e.g., antibodies or antibody fragments). Thecomposition further includes a competitor affinity reagent 1630 (e.g.,aptamers) that is free in solution due to being separate from the probe1610. The competitor species 1630 are able to freely associate with abinding partner, epitope or target moiety, but the competitor speciesare separable and can dissociate from the binding partner, epitope, ortarget moiety. FIG. 16B illustrates a detectable probe compositionincluding a competitor component that is attached to the detectableprobe. The detectable probe 1610 includes a plurality of attachedbinding components 1620 (e.g., antibodies or antibody fragments) and aplurality of attached competitor components 1630 (e.g., aptamers). Adetectable probe or affinity reagent composition including an attachedcompetitor component may be advantageous for facilitating dissociationof a bound affinity reagent with a slow off-rate due to a net decreasein likelihood of the strongest binder being bonded to a target at anygiven time.

Alternatively, a competitor species may include a species that competeswith a binding partner, epitope, or target moiety to bind the detectableprobe or affinity reagent. For example, a competitor species may be ashort peptide sequence (e.g., 2-15 amino acid residues) containing atarget sequence that is free in solution. The free peptide may be ableto competitively bind with a detectable probe or affinity reagent,thereby altering the apparent avidity of a detectable probe or affinityreagent in an analogous fashion to a competitor for binding interactionswith a binding partner, epitope, or target moiety.

A competitor species may be present when a detectable probe or affinityreagent is contacted with a binding partner, epitope, or target moiety.A competitor species may be introduced before or after a detectableprobe or affinity reagent is contacted with a binding partner, epitope,or target moiety. The ratio of detectable probe or affinity reagent tocompetitor species in a composition may be adjusted to tailor theavidity characteristics of the composition. A detectable probe oraffinity reagent composition may be adjusted to tailor a bindingcharacteristic, such as probe on-rate or off-rate, to achieve a desiredlevel of avidity. A competitor species may be present in a ratio of atleast about 1:1000000, 1:100000, 1:10000, 1:1000, 1:100, 1:10, 1:5, 1:2,1:1, 2:1, 5:1, 10:1, 100:1, 1000:1, 10000:1, 100000:1, 1000000:1 or morerelative to the detectable probe or affinity reagent, on a mass or molarbasis. Alternatively or additionally, a competitor species may bepresent in a ratio of no more than about 1000000:1, 100000:1, 10000:1,1000:1, 100:1, 10:1, 5:1, 2:1, 1:1, 1:2, 1:5, 1:10, 1:100, 1:1000,1:10000, 1:100000, 1:1000000 or less relative to the detectable probe oraffinity reagent, on a mass or molar basis.

The presence of a competitor species, for example, in the form of acompetitor component, in a detectable probe or affinity reagentcomposition, may affect a binding or affinity characteristic of thedetectable probe or affinity reagent. The presence of a competitorspecies, for example, in the form of a competitor component, in adetectable probe or affinity reagent composition may affect an off-rate,on-rate, dissociation constant, or avidity, of the detectable probe oraffinity reagent. The presence of a competitor species, for example, inthe form of a competitor component, in a detectable probe or affinityreagent composition may increase an off-rate, on-rate, dissociationconstant, or avidity, of the detectable probe or affinity reagent. Thepresence of a competitor species, for example, in the form of acompetitor component, in a detectable probe or affinity reagentcomposition may decrease an off-rate, on-rate, dissociation constant, oravidity, of the detectable probe or affinity reagent. The presence of acompetitor species, for example, in the form of a competitor component,may increase or decrease an off-rate, on-rate, dissociation constant, oravidity of the detectable probe or affinity reagent by a factor of atleast about 2, 5, 10, 25, 50, 100, 250, 500, 1000, 5000, 10000, 50000,100000, 500000, 1000000, or more. Alternatively or additionally, thepresence of a competitor species, for example, in the form of acompetitor component, may increase or decrease an off-rate, on-rate,dissociation constant, or avidity of a detectable probe or affinityreagent by a factor of no more than about 1000000, 500000, 100000,50000, 10000, 5000, 1000, 500, 250, 100, 50, 25, 10, 5, 2, or less.

A detectable probe or affinity reagent composition may include one ormore components that are configured to alter the binding characteristicsof the detectable probe or affinity reagent. A component may be added toa detectable probe or affinity reagent composition to increase theon-rate, off-rate, dissociation constant, and/or avidity of thedetectable probe or affinity reagent composition. A component may beadded to a detectable probe or affinity reagent composition to decreasethe on-rate, off-rate, dissociation constant, and/or avidity of thedetectable probe or affinity reagent composition. A component may affectthe affinity or avidity of a detectable probe or affinity reagent bychemically altering a binding interaction between the detectable probeor affinity reagent and a binding partner, epitope, or target moiety(e.g., altering the conformation of a binding component, weakening theelectrostatic interaction between a binding component and a bindingpartner). A component may affect the affinity or avidity of a detectableprobe or affinity reagent by creating a weak secondary bindinginteraction with the detectable probe or affinity reagent.

A component may be added to a detectable probe or affinity reagentcomposition to alter the binding interaction between the detectableprobe or affinity reagent composition and a binding partner, epitope, ortarget moiety. The added component may alter a binding interaction byaltering the binding partner, epitope, or target moiety, altering thedetectable probe or affinity reagent, or by altering the bindinginteraction between the detectable probe or affinity reagent compositionand the binding partner, epitope, or target moiety. An added componentmay cause a conformational change in a detectable probe or affinityreagent, binding component, and/or binding partner, epitope, or targetmoiety that increases or decreases the likelihood and/or strength of abinding interaction. An added component may alter a binding interaction,for example by electrostatically screening a binding interaction orcompeting to form a binding interaction.

A denaturant, surfactant, or chaotropic agent may be added to adetectable probe or affinity reagent composition to alter a bindinginteraction between the detectable probe or affinity reagent and abinding partner, epitope, or target moiety. A denaturant, surfactant, orchaotropic agent may be added to a detectable probe or affinity reagentcomposition to alter a binding characteristic of the detectable probe oraffinity reagent, such as an on-rate, off-rate, dissociation constant,or avidity. Denaturants, surfactants, or chaotropic agents mayfacilitate the removal of a detectable probe or affinity reagent from abinding partner, epitope, or target moiety. Denaturants, surfactants, orchaotropic agents may be introduced to a detectable probe or affinityreagent composition after probe binding to facilitate the removal of thedetectable probe or affinity reagent from a binding partner, epitope, ortarget moiety. Binding of a detectable probe or affinity reagent in thepresence of a denaturant, surfactant, or chaotropic agent may reduce theon-rate of the detectable probe or affinity reagent when binding to abinding partner, epitope, or target moiety. The concentration of adenaturant, surfactant, or chaotropic agent in a detectable probe oraffinity reagent composition may be adjusted to tune an affinitycharacteristic of the detectable probe or affinity reagent. Theconcentration of a denaturant, surfactant, or chaotropic agent in adetectable probe or affinity reagent composition may be limited to avoiddestabilizing the detectable probe or affinity reagent or a component ofthe detectable probe or affinity reagent (e.g., a DNA origamicomponent). A denaturant, surfactant, or chaotropic agent in adetectable probe or affinity reagent composition may be combined withheat to facilitate the removal of the detectable probe or affinityreagent from a binding partner, epitope, or target moiety.

A salt, such as a metal salt, may be added to a detectable probe oraffinity reagent composition to alter a binding interaction between thedetectable probe or affinity reagent and a binding partner, epitope, ortarget moiety. A salt, or the ionic species including a salt, may forminteractions with a detectable probe or affinity reagent or a bindingpartner, epitope, or target moiety that alter the chemistry of thebinding interaction between the detectable probe or affinity reagent anda binding partner, epitope, or target moiety. A salt, or an ionicspecies including a salt, may disrupt the chemistry of the bindinginteraction between a detectable probe or affinity reagent and a bindingpartner, epitope, or target moiety. A salt, or an ionic speciesincluding a salt, may facilitate a binding interaction between adetectable probe or affinity reagent and a binding partner, epitope, ortarget moiety. In some configurations, a salt may be added to adetectable probe or affinity reagent composition to facilitate theremoval of the detectable probe or affinity reagent from a bindingpartner, epitope, or target moiety. A salt in a detectable probe oraffinity reagent composition may be combined with heat to facilitate theremoval of the detectable probe or affinity reagent from a bindingpartner, epitope, or target moiety. A salt may be introduced to adetectable probe or affinity reagent composition after probe binding toalter a binding interaction of the detectable probe or affinity reagentwith a binding partner, epitope, or target moiety (e.g., to remove adetectable probe or affinity reagent).

A detectable probe or affinity reagent composition may further include abinding molecule that alters the avidity and/or observability of thedetectable probe or affinity reagent. A binding molecule may include amolecule that is configured to form a reversible or irreversible bindinginteraction with a detectable probe or affinity reagent. A bindingmolecule may form a weak binding interaction with a binding partner,epitope, target moiety, or a detectable probe or affinity reagent tokeep the detectable probe or affinity reagent near a possible target,thereby increasing the likelihood that a binding interaction may occur.A binding molecule may include additional label components that increasethe signal produced when a binding interaction occurs. Exemplary bindingmolecules are described elsewhere herein. A binding molecule may beprovided to a detectable probe or affinity reagent composition before,during, or after the detectable probe or affinity reagent has beencontacted with a binding partner, epitope, or target moiety.

A detectable probe or affinity reagent composition of the presentdisclosure can be provided in kit form including, if desired, a suitablepackaging material. In one embodiment, for example, a detectable probeor affinity reagent composition can include a plurality of detectableprobes or affinity reagents, for example, provided in a solution orsuspension. In another embodiment, for example, a detectable probe oraffinity reagent composition can include a plurality of detectableprobes or affinity reagents, for example, provided as a solid, such ascrystals or a lyophilized solid. Accordingly, any combination ofreagents or components that is useful in a method of the disclosure,such as those set forth herein previously in regard to particularmethods, can be included in a kit provided by the disclosure. Withoutlimitation, those reagents or components may include buffers, salts,stabilizers, retaining components, binding components, label components,and competitor affinity reagents. For example, a kit can include aplurality of detectable probes or affinity reagents provided in astorage buffer containing a stabilizing surfactant and an oxygenscavenger.

As used herein, the phrase “packaging material” refers to one or morephysical structures used to house the contents of the kit, such asdetectable probes, affinity reagents or the like. The packaging materialcan be constructed by well-known methods, preferably to provide asterile, contaminant-free environment. The packaging materials employedherein can include, for example, those customarily utilized in affinityreagent systems. Exemplary packaging materials include, withoutlimitation, glass, plastic, paper, foil, and the like, capable ofholding within fixed limits a component useful in the methods of thedisclosure such as a detectable probe or affinity reagent composition.

The packaging material can include a label which indicates that thedetectable probes or affinity reagents can be used for a particularmethod. For example, a label can indicate that the kit is useful fordetecting a particular binding partner, epitope, or target moiety,thereby providing a characterization during a polypeptide assay. Inanother example, a label can indicate that the kit is useful for atherapeutic or diagnostic purpose.

Instructions for use of the packaged reagents or components are alsotypically included in a kit of the disclosure. “Instructions for use”typically include a tangible expression describing the reagent orcomponent concentration or at least one assay method parameter, such asthe relative amounts of kit components and sample to be admixed,maintenance time periods for reagent/sample admixtures, temperature,buffer conditions, and the like.

A kit or composition can include a plurality of different detectableprobes and/or different affinity reagents that differ with respect tothe number or type of binding component(s) attached thereto.Alternatively or additionally, a kit or composition can include aplurality of different detectable probes and/or different affinityreagents that are attached to different label components, respectively.The detectable probes or affinity reagents, although differing withrespect to the number or type of binding components or with respect tothe number or type of label components, can nonetheless havesubstantially the same type of retaining component. For example, aplurality of different detectable probes and/or different affinityreagents can contain nucleic acid origami-based retaining componentswherein the structure of the origami or the structure of a scaffold inthe origami is the same for all the different probes and/or agents. Aplurality of different detectable probes and/or different affinityreagents can include at least about 2, 3, 4, 5, 10, 25, 50, 100, 250,500, 1000 or more different detectable probes and/or different affinityreagents. Alternatively or additionally, a plurality of differentdetectable probes and/or different affinity reagents can include at mostabout 1000, 500, 250, 100, 50, 25, 10, 5, 4, 3, 2 or fewer detectableprobes and/or different affinity reagents.

Methods of Use

Detectable probes or affinity reagents of the present disclosure may beutilized for a broad range of uses, including binding assays. Ingeneral, utilization of detectable probes or affinity reagentcompositions may involve one or more of the steps of: 1) contacting adetectable probe or affinity reagent with a solution and/or solidsupport having a binding partner for the detectable probe or affinityreagent; 2) permitting the detectable probe or affinity reagent to bindwith the binding partner in the solution or on the solid support; 3)rinsing unbound probes from the solution and/or solid support; 4)observing the solution and/or solid support to detect a signal from oneor more detectable probes or affinity reagents; 5) removing one or morebound probes or reagents from the binding partner; 6) optionallyrepeating one or more of steps 1)-5); and 7) using the presence and/orabsence of a signal from one or more probes or reagents to predict thepresence and/or absence of the binding partner in the solution and/or onthe solid support.

A method of observing binding interactions between detectable probes oraffinity reagents and binding partners may be utilized for any of avariety of characterizations. In some cases, a detectable probe oraffinity reagent may be utilized to facilitate a polypeptidecharacterization assay. The assay may be configured to determine orpredict one or more characteristics (e.g., size, identity, isotype,etc.) for one or more polypeptides. Alternatively or additionally, theassay may be an assay configured to determine or predict the amount ofone or more polypeptides in a sample. A polypeptide characterizationassay or quantification assay may be configured for single-moleculedetection where one or more polypeptide molecules are individuallyresolved. For example, in a multiplex format one or more characteristicand/or quantity can be predicted or determined for each polypeptide of aplurality of polypeptides based on detection of the polypeptidesindividually. In some configurations, a polypeptide assay may includesingle-molecule characterization or quantification of polypeptides todetermine the presence of one or more epitopes (e.g., dimer, trimer, ortetramer amino acid sequences; post-translational modifications, etc.)in a plurality of polypeptides. Detectable probes or affinity reagentsmay be advantageous for polypeptide characterization assays due to theirstrong avidity for binding targets and their strong signal output,thereby giving high-confidence binding interaction data.

A polypeptide or other analyte can be attached to a structured nucleicacid particle (SNAP) when bound to an affinity reagent or detectableprobe. FIG. 58A shows polypeptide 5840 attached to SNAP 5850.Polypeptide 5840 is an exemplary analyte and can be replaced with otheranalytes of interest. SNAP 5850 functions as a retaining component forpolypeptide 5840 and can be replaced or modified with a retainingcomponent set forth herein in the context of affinity reagents anddetectable probes. For example, SNAP 5850 can be a nucleic acid origamior can be replaced with a fluorescent particle. Polypeptide 5840 can becovalently or non-covalently attached to SNAP 5850. Polypeptide 5840 canbe contacted with a detectable probe having a SNAP-based retainingcomponent 5810, one or more label components 5820 and one or morebinding components 5830. Optionally, SNAP 5810 can be replaced ormodified with a retaining component set forth herein in the context ofaffinity reagents and detectable probes. For example, SNAP 5810 can be anucleic acid origami or can be replaced with a fluorescent particle.Because polypeptide 5840 is a binding partner for the one or morebinding components 5830, a complex is formed. The complex includes SNAP5850 which is attached to polypeptide 5840 and SNAP 5810 which isattached to one or more binding components 5830, at least one of whichis bound to polypeptide 5840, SNAP 5810 also being attached to at leastone label component 5820. Optionally, SNAP 5850 can be attached to asolid support such as a site on an array. A SNAP can be attached to asolid support using any of a variety of covalent or non-covalentchemistries including, but not limited to those set forth herein in thecontext of attaching components of affinity reagents or detectableprobes to each other.

In some configurations, a polypeptide assay may include contacting adetectable probe or affinity reagent with a plurality of polypeptides,wherein each polypeptide of the plurality of polypeptides is bound to asolid support at a unique, optically observable spatial location, suchas a site in an array of polypeptides. The detection of a signal at agiven spatial location may be evidence that a detectable probe oraffinity reagent has bound to a polypeptide at the spatial location,thereby suggesting the presence of a particular epitope or target moietyin the polypeptide at the spatial location. The predicted or determinedpresence or absence of one or more epitopes or target moieties maypredict or determine a characteristic for a polypeptide bound at a givenspatial location. Moreover, the quantity of a particular polypeptide ina sample can be predicted or determined based on the intensity of signalfrom a given spatial location and/or based on the number of sites in anarray that produce a signal indicating that a particular probe (orseries of probes) has bound.

A multiplex binding reaction is shown in FIG. 58B. The reaction isexemplified for polypeptide analytes but can be performed with otheranalytes known in the art or set forth herein. Polypeptide array 5800includes four different polypeptides 5841 through 5844. Each of thepolypeptides is attached to a SNAP 5851. Attachment can be covalent ornon-covalent. Array 5800 is contacted with a plurality of detectableprobes. Each of the probes includes a SNAP-based retaining component5811 attached to at least one label component and at least one bindingcomponent. Three different detectable probes are shown including a firstprobe having SNAP 5811 attached to at least one label moiety 5821 and atleast one binding moiety 5831, a second probe having SNAP 5811 attachedto at least one label moiety 5822 and at least one binding moiety 5832,and a third probe having SNAP 5811 attached to at least one label moiety5823 and at least one binding moiety 5833. The product of the bindingreaction is binding of the first probe and third probe to respectivepolypeptides on array 5800. Because polypeptide 5841 is a bindingpartner for the one or more binding components 5831, a first complex isformed. The first complex includes SNAP 5851 which is attached topolypeptide 5841 and SNAP 5811 which is attached to one or more bindingcomponents 5831, at least one of which is bound to polypeptide 5841,SNAP 5811 also being attached to at least one label component 5821.Because polypeptide 5843 is a binding partner for the one or morebinding components 5833, a second complex is formed on array 5800. Thefirst complex includes SNAP 5851 which is attached to polypeptide 5843and SNAP 5811 which is attached to one or more binding components 5833,at least one of which is bound to polypeptide 5843, SNAP 5811 also beingattached to at least one label component 5823. In this example,polypeptides 5842 and 5844 remain unbound since they are not bindingpartners with any of the detectable probes. Also the third detectableprobe, which includes SNAP 5811 attached to at least one bindingcomponent 5832 and at least one label component 5822, does not have abinding partner on array 5800 and remains unbound. The first and seconddetectable probes can be detected on the array where they are spatiallyresolved and where they can be further distinguished and identifiedbased on their different label types. Accordingly, signals detected fromthe array sites can be used to identify the polypeptides based onknowledge of the binding properties for each detectable probe and thelabel type associated with each detectable probe.

In the example of FIG. 58B, the polypeptides although separated intoindividual sites on array 5800, are attached to SNAPs having a commonstructure. For example, SNAP 5851 can be a nucleic acid origami and thepolypeptides can be attached to origami having the same scaffold fold aseach other. A subset of one or more staples in the origami can differbetween the origamis, for example, to accommodate attachment of thedifferent polypeptides. The use of a common SNAP structure, or otherretaining component for that matter, can provide convenient loading ofthe polypeptides on the array. For example, the array can be configuredto have uniform sites that interact with uniform structural elements ofthe SNAPs to achieve attachment of the polypeptides to the array. Acommon retaining component structure can also be used for a plurality ofdifferent detectable probes or a plurality of different affinityreagents. As exemplified in FIG. 58B, the three detectable probes differwith respect to the label components and binding components that areattached to SNAP 5811. However, the three affinity probes havesubstantially the same SNAP structure. For example, SNAP 5811 can be anucleic acid origami having the same scaffold fold for each of the threedetectable probes. A subset of one or more staples in the origami candiffer between the different detectable probes, for example, toaccommodate attachment of different label components or differentbinding components. Although FIG. 58B is exemplified with common SNAPsattached to different analytes and common SNAPs attached to differentdetectable probes, it will be understood that the SNAPs can differ instructure instead. Moreover, the SNAPs can include sequence tags thatdiffer from one detectable probe to another or that differ from onepolypeptide to another. The tags can be used to identify individualprobes in a mixture or to identify individual polypeptides in an array.

Arrays can be used for multiplex processing of analytes, whereby themultiple different types of analytes are manipulated or detected inparallel. Particularly useful analytes include, but are not limited to,binding partners for affinity reagents set forth herein, such aspolypeptides, nucleic acids, or the like. Although it is also possibleto serially process different types of analytes using one or more stepsof the methods set forth herein, parallel processing can provide costsavings, time savings and uniformity of conditions. An array can includeat least 2, 10, 100, 1000, 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, ormore different analyte sites. Alternatively or additionally, an arraycan include at most 1×10⁹, 1×10⁸, 1×10⁷, 1×10⁶, 1×10⁵, 1×10⁴, 1000, 100,10, 2 or fewer, different analyte sites. The different analytes mayoptionally be attached to the sites via structured nucleic acidparticles or retaining components that have common structures. As such,sites of an array can be attached to structured nucleic acid particlesor retaining components having substantially the same structure as eachother, and each of the structured nucleic acid particles or retainingcomponents in the array can be attached to a different analyte.

An array can be attached to an inner surface of a flow cell wall or to asolid support inside of a flow cell. The flow cell or solid support canbe made from any of a variety of materials used for analyticalbiochemistry. Suitable materials may include glass, polymeric materials,silicon, quartz (fused silica), borofloat glass, silica, silica-basedmaterials, carbon, metals, an optical fiber or bundle of optical fibers,sapphire, or plastic materials. The material can be selected based onproperties desired for a particular use. For example, materials that aretransparent to a desired wavelength of radiation are useful foranalytical techniques that will utilize radiation at that wavelength.Conversely, it may be desirable to select a material that does not passradiation of a certain wavelength (e.g. being opaque, absorptive orreflective). Other properties of a material that can be exploited areinertness or reactivity to certain reagents used in a downstreamprocess, such as those set forth herein, or ease of manipulation, or lowcost of manufacture.

A polypeptide or other analyte can be attached to a support in a waythat provides detection at a single molecule level. For example, aplurality of different polypeptides can be attached to a solid supportin a way that an individual detectable probe or affinity reagent thatbinds to an individual polypeptide site on the support can bedistinguished from all neighboring sites on the array even if theneighboring sites bind to detectable probes or affinity reagents. Assuch, one or more different polypeptides (or other binding partners fordetectable probes or affinity reagents set forth herein) can be attachedto a solid support in a format where each single polypeptide molecule isphysically isolated and detected in a way that the single molecule isresolved from all other molecules on the solid support. Alternatively, amethod of the present disclosure can be carried out for one or moreensembles, an ensemble being a population of analytes of substantiallythe same type such as a population of nucleic acids or polypeptideshaving a common sequence.

FIG. 22 illustrates a configuration of a polypeptide assay. A pluralityof polypeptides is bound to a solid support 2210, optionally byanchoring groups 2230 (e.g. SNAP or chemical linker). Detectable probes2240 are contacted with the plurality of polypeptides, permittingdetectable probes 2240 to bind with available binding targets. After thecontacting, detectable probes 2240 have bound to the first polypeptide2221 and the fourth polypeptide 2224, and have not bound to the secondpolypeptide 2222 or the third polypeptide 2223. If observed by anappropriate detection system, such as a fluorescent microscope, adetectable signal may be observed at spatial locations on the solidsupport 2210 corresponding to the location of the first polypeptide 2221and the fourth polypeptide 2224.

Polypeptides may be of natural or synthetic origin. Polypeptides maycontain one or more post-translational modifications. Alternatively oradditionally, one or more post-translational modifications can be absentfrom a polypeptide. In some cases, polypeptides may be treated toremove, reverse or alter post-translational modifications. For example,a polypeptide assay set forth herein can be performed to detect one ormore polypeptides before and after removing, reversing or alteringpost-translational modifications. Comparing the before and after resultscan provide benefits such as increased confidence in identifying thenumber or type of post-translational modifications for a polypeptide. Insome cases, polypeptides may be treated to produce post-translationallymodified. For example, a polypeptide assay set forth herein can beperformed to detect one or more polypeptides before and after makingpost-translational modifications. Conversely, a polypeptide assay setforth herein can be performed to detect one or more polypeptides beforeand after removing post-translational modifications. Comparing thebefore and after results can provide benefits such as identifying thenumber or type of post-translational modifications or post-synthesismodifications to which a polypeptide is susceptible. In some cases,polypeptides may be proteolyzed to remove at least a portion of thepolypeptide. For example, a polypeptide assay set forth herein can beperformed to detect one or more polypeptides before and afterproteolysis. Comparing the before and after results can provide benefitssuch as locating an epitope or target moiety in a polypeptide relativeto the location of proteolytic site in the polypeptide. A furtherbenefit of comparing the before and after results can be easieridentification of the number or type of post-translational modificationsin a polypeptide that is susceptible to proteolysis.

Post-translational modifications may include myristoylation,palmitoylation, isoprenylation, prenylation, farnesylation,geranylgeranylation, lipoylation, flavin moiety attachment, Heme Cattachment, phosphopantetheinylation, retinylidene Schiff baseformation, dipthamide formation, ethanolamine phosphoglycerolattachment, hypusine, beta-Lysine addition, acylation, acetylation,deacetylation, formylation, alkylation, methylation, C-terminusamidation, arginylation, polyglutamylation, polyglyclyation,butyrylation, gamma-carboxylation, glycosylation, glycation,polysialylation, malonylation, hydroxylation, iodination, nucleotideaddition, phosphoate ester formation, phosphoramidate formation,phosphorylation, adenylylation, uridylylation, propionylation,pyrolglutamate formation, S-glutathionylation, S-nitrosylation,S-sulfenylation, S-sulfinylation, S-sulfonylation, succinylation,sulfation, glycation, carbamylation, carbonylation, isopeptide bondformation, biotinylation, carbamylation, oxidation, reduction,pegylation, ISGylation, SUMOylation, ubiquitination, neddylation,pupylation, citrullination, deamidation, elminylation, disulfide bridgeformation, proteolytic cleavage, isoaspartate formation, racemization,and protein splicing. A particularly interesting and usefulpost-translational modification is proteolysis which may besite-specific (i.e. occurring at particular amino acid residues or aminoacid sequences) or non-site specific. Post-translational modificationscan be reversed or removed from a polypeptide using biological enzymes,chemical agents, or physical manipulations such as those known to thoseskilled in the art. Polypeptides can be modified post-translationallyusing biological enzymes, chemical agents, or physical manipulationssuch as those known to those skilled in the art.

Characterized polypeptides may be of a particular size. A polypeptidemay be at least about 0.1 Daltons (Da), 0.5 Da, 1 Da, 5 Da, 10 Da, 50Da, 100 Da, 200 Da, 300 Da, 400 Da, 500 Da, 600 Da, 700 Da, 800 Da, 900Da, 1 kiloDalton (kDa), 1.5 kDa, 2 kDa, 2.5 kDa, 3 kDa, 3.5 kDa, 4 kDa,4.5 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, 15 kDa, 20 kDa, 25kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa,200 kDa, 300 kDa, 400 kDa, 500 kDa, 600 kDa, 700 kDa, 800 kDa, 900 kDa,1000 kDa, 1200 kDa, 1400 kDa, 1600 kDa, 1800 kDa, 2000 kDa, 2500 kDa,3000 kDa, 3500 kDa, 4000 kDa, or more. Alternatively or additionally, apolypeptide may be no more than about 4000 kDa, 3500 kDa, 3000 kDa, 2500kDa, 2000 kDa, 1800 kDa, 1600 kDa, 1400 kDa, 1200 kDa, 1000 kDa, 900kDa, 800 kDa, 700 kDa, 600 kDa, 500 kDa, 400 kDa, 300 kDa, 200 kDa, 100kDa, 90 kDa, 80 kDa, 70 kDa, 60 kDa, 50 kDa, 40 kDa, 30 kDa, 25 kDa, 20kDa, 15 kDa, 10 kDa, 9 kDa, 8 kDa, 7 kDa, 6 kDa, 5 kDa, 4.5 kDa, 4 kDa,3.5 kDa, 3 kDa, 2.5 kDa, 2 kDa, 1.5 kDa, 1 kDa, 900 Da, 800 Da, 700 Da,600 Da, 500 Da, 400 Da, 300 Da, 200 Da, 100 Da, 50 Da, 10 Da, 5 Da, 1Da, 0.5 Da, or less. A polypeptide may contain a minimum or maximumnumber of amino acid residues. A polypeptide may contain at least about2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80,90, 100, 125, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000,1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000,20000, 30000, 40000 or more amino acid residues. Alternatively oradditionally, a polypeptide may contain no more than about 40000, 30000,20000, 15000, 10000, 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2000,1500, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 250, 200, 150, 125,100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5,4, 3, or less amino acid residues.

A polypeptide assay may involve one or more fluid transfer operations,including fluids containing detectable probes or affinity reagents, andfluids that mediate binding interactions involving detectable probe oraffinity reagents (e.g., rinse fluids, removal reagents). A fluidtransfer operation may occur when a fluid is transferred into ortransferred out of a fluidic device, such as a flow cell or chip. Apolypeptide characterization assay may require that a plurality ofpolypeptides bound to a solid support be in contact with a fluid mediumat all time. Fluid transfer operations may utilize a second fluid mediumto displace a first fluid medium from a flow cell to ensure the presenceof fluid in contact with the solid support including a plurality ofpolypeptides at all times.

A sample may be provided to a polypeptide detection system or method. Asample may be provided as a fraction, for example, including a pluralityof polypeptides or other analytes. A sample may be provided as afraction including a plurality of polypeptide conjugates or otheranalyte conjugates. An analyte conjugate may include the analyte (e.g. apolypeptide analyte) attached to a solid support directly of via alinker moiety such as a structured nucleic acid particle, polymer, orprotein moiety. A sample may be provided to a detection system or methodin a liquid medium (e.g., an aqueous, pH buffering medium). A sample maybe provided to a detection system or method as a solid (e.g., alyophilized, precipitated, or crystallized sample). A solid may bedissolved or suspended in a liquid medium before, during or after beingadded to a detection system or method. A sample may be stored in adetection system after being provided to the system.

A sample including polypeptides or other analytes may be bound to asolid support in a fluidic device. A fluid including the sample may bepumped or flowed through a fluidics system to a detection chamber, suchas a flow cell or well, including a solid support within a fluidicsdevice. The fluid including the sample may be contacted to the solidsupport for a sufficient amount of time to attach the analytes to thesolid support. Optionally the analytes can be conjugated to a substance,such as a structured nucleic acid particle, that mediates attachment ofthe analyte to the solid support. In some cases, additional fluids maybe mixed with sample fluid to facilitate the binding of the analytes tothe solid support. If a sample includes polypeptides, a solid supportmay first be contacted with a fluid including polypeptide linking groups(e.g., structured nucleic acid particles, polymers, proteins) for asufficient amount of time to deposit the polypeptide linking groups onthe surface of the solid support. After the solid support has beendeposited with polypeptide linking groups, a fluid medium including thesample may be pumped or flowed into the detection chamber containing thesolid support in the fluidic device. An attachment reaction between thepolypeptides and the polypeptide linking groups may be allowed to occurfor a sufficient amount of time to attach the polypeptides to thelinking groups. One or more reagents may be added to the fluidic deviceto facilitate a polypeptide attachment reaction, such as reactantsand/or catalysts. In some cases, polypeptides and polypeptide linkinggroups may be configured to react via a spontaneous attachment reaction,such as a “click” reaction. In some cases, polypeptides may be directlyattached to a solid support, for example by a bond between a functionalgroup on the polypeptide and a functional group on the solid support.After an attachment reaction, the solid support will include one or morepolypeptides bound to the solid support.

After analytes (e.g. polypeptides) have been attached to a solidsupport, for example within a fluidic device, the detection chamber ofthe fluidic device may be rinsed one or more times. A rinsing processmay utilize one or more washing or rinsing reagents that are pumped orflowed into the fluidic device, either together or in succession. Avolume of rinsing fluid provided to the fluidic device may exceed thetotal volume of the detection chamber including the solid support. Therinsing step may remove some or all unbound polypeptides, polypeptideconjugates, or other reagents. The flow rate of rinsing fluid to thefluidic device may be limited to prevent the dislodging of attachedanalytes from the solid support.

Optionally, attached analytes (e.g. conjugated polypeptides) may besubjected to one or more processes that alter the structure of theanalytes. For example polypeptide analytes may be partially or fullydenatured, for example, by the addition of a denaturing fluid to thefluidic device. A denaturing fluid may be contacted to the solid supportfor a sufficient time to promote full denaturation of a polypeptide, ormay be briefly contacted to the solid support to cause partialdenaturation of a polypeptide. Polypeptides may also be altered by theapplication of altering reagents to the solid support, for exampleenzymes (ligases, kinases, glycosylases, phosphatases, proteases, etc.),oxidants, or reductants. A fluidic device may undergo a rinsing processafter the addition of a structure-altering reagent, thereby removingresidual reagent from the fluidic device. In some cases, the solidsupport may be held in contact with an altering agent (e.g., adenaturant) until an affinity reagent is introduced to the fluidicdevice. In some cases, a polypeptide may partially or fully re-foldafter a structure-altering processes.

An affinity reagent may be contacted with a solid support including aplurality of polypeptides (e.g. an array of polypeptides) or otheranalytes, for example, in a fluidic device. In some configurations, anaffinity reagent may be provided as a detectable probe or affinityreagent. Affinity reagents such as detectable probes may be provided ina fluidic medium that is pumped or flowed into a fluidic device. Afluidic medium may include a single species of affinity reagent ordetectable probe, or may include a mixture of differing species ofaffinity reagents or detectable probes. For example, a fluidic mediummay include at least two detectable probes or affinity reagents withdiffering epitope-binding specificities. Or, a fluidic medium mayinclude at least two detectable probes or affinity reagents that aredifferent types of detectable probes or affinity reagents, e.g., 1aptamer probe and 1 antibody probe. Affinity reagents or detectableprobes may be contacted to a solid support in a fluidic device for asufficient amount of time to promote binding of an affinity reagent or adetectable probe to an epitope or target moiety that is present in atleast one analyte (e.g. an amino acid sequence epitope in apolypeptide).

Affinity reagents or binding components that are utilized in a bindingassay may be distinguished by the nature of their binding specificity.In particular, an affinity reagent may have an epitope bindingspecificity that has been characterized in a probabilistic fashion.Rather than being characterized in a binary fashion (e.g., affinityreagent A binds to epitope X, does not bind to epitope Y), an affinityreagent of the present disclosure may be characterized by a plurality ofbinding probabilities. For example, over the 8000 possible trimer aminoacid sequences (20×20×20), a non-zero binding probability for anaffinity reagent may be known, measured, or estimated for some or all ofthe 8000 sequences. In some cases, an affinity reagent may be useful tothe present disclosure if it has a high known, measured, or estimatedbinding probability for a first epitope (e.g., greater than 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.9%, 99.99%, 99.999% orhigher probability of binding), and a low known, measured, or estimatedbinding probability for a second epitope (e.g., less than 1%, 0.1%,0.01%, 0.001%, 0.0001%, 0.00001%, 0.000001% or lower probability ofbinding). In some cases, an affinity reagent may include high known,measured, or estimated binding probabilities for a family of epitopes(e.g., greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,99%, 99.9%, 99.99%, 99.999% or higher probability of binding), and lowknown, measured, or estimated binding probabilities for other epitopes(e.g., less than 1%, 0.1%, 0.01%, 0.001%, 0.0001%, 0.00001%, 0.000001%or lower probability of binding). Families of epitopes may includeepitopes related by amino acid sequence (e.g., amino acid sequences AXA,where A is alanine and X may be any amino acid; amino acid sequencesαAAAβ, where α and β are independently any possible amino acid flankingsequences) or may be related by chemical properties (e.g., non-polar,polar, positively charged, negatively charged, post-translationalmodifications, etc.).

After an affinity reagent, such as a detectable probe, has been givensufficient time to bind to an analyte (e.g. a polypeptide analyte) in afluidic device, a detection chamber of the device may undergo one ormore washes or rinses to remove any unbound affinity reagents. Apolypeptide characterization assay may only utilize a single rinse stepafter affinity reagent binding to minimize the likelihood of ananalyte-affinity reagent interaction becoming disrupted. An affinityreagent rinse fluid may be displaced from a fluidic device after arinsing process by a medium that is configured for performing physicalmeasurement, such as an optical imaging buffer.

An analyte:affinity reagent or analyte:probe interaction may be measuredafter affinity reagents or detectable probes have been contacted with aplurality of analytes bound to a solid support. Physical measurements,such as detection of label components of detectable probes, may occurunder quiescent fluid conditions. A fluid may be held static in a fluidby isolating all inlet and outlet ports, for example by the closing ofvalves. A fluid may be held in the fluidic device for a sufficientamount of time to permit imaging of one, some or all unique, opticallyobservable spatial locations.

After binding and/or physical measurements of analyte:affinity reagentor analyte:probe interactions have occurred, bound affinity reagents ordetectable probes may be stripped from the analytes by the addition of adisrupting medium to the fluidic device. The disrupting medium mayinclude a denaturant, chaotrope, or other chemical entity that is likelyto disrupt an analyte: affinity reagent or analyte:probe interaction.The disrupting medium may be contacted with the analytes under quiescentor flowing conditions. After a disrupting medium has been contacted withthe analytes, the fluidic device may be rinsed or washed with a rinsingmedium one or more times to remove any unbound affinity reagents orunbound detectable probes from the fluidic device.

In some cases, analytes (e.g. polypeptide analytes) may be removed fromthe surface of a solid support during or after a binding measurement.For example, a plurality of polypeptides may be characterized todetermine which polypeptides are glycosylated, followed by the releasefrom the solid support of all non-glycosylated polypeptides. In somecases, all analytes may be released from the solid support after acharacterization assay is complete to permit reuse of the fluidicdevice. An analyte or analyte-conjugate may be released from a solidsupport by the addition of a stripping fluid. The stripping fluid mayprovide a more stringent wash than other rinse mediums utilized duringthe characterization assay such that it causes displacement of analytesfrom the solid support. Displacement of analytes from a solid supportmay also utilize physical conditions such as heat or light, for example,by severing a photoactivatable linker between an analyte and solidsupport.

During or following the displacement of analytes from a solid support, arinsing medium may flow through the fluidic device to remove displacedanalytes. A solid support may be subsequently observed by a physicalmeasurement method to determine or confirm the displacement of analytesfrom the surface.

Regardless of the specificities described above, in some cases, one mayidentify a particular polypeptide analyte based upon a series ofobservations of different probes that either bind or don't bind to thatprotein. Such series may be evaluated using software algorithms thatutilize probabilistic modeling to evaluate probabilities of given seriesbeing identifying of given proteins or polypeptides. In a multiplexedconfiguration, a plurality of different polypeptides can be observed fora pattern of binding and non-binding. For example, the proteins can bedisplayed in an array, the array composed of individually resolvablesites, each site having one of the polypeptides. A series ofobservations can be made for each site as to whether different probeseither bind or don't bind. The identity of the polypeptide at each sitecan be determined from the series of observations at each site. Theoverall pattern for the series of observations at each site can beuseful for identifying multiple polypeptides across the array. Examplesof software algorithms, methods and compositions that are useful foridentifying proteins are described, for example, in publishedInternational Patent Application No. WO 2019/133892, U.S. Pat. No.10,473,654, or US Pat. App. Pub. No. 2020/0286584 A1, each of which isincorporated herein by reference.

In some cases, detection, identification or characterization ofpolypeptides may utilize affinity reagents set forth herein. Affinityreagents of the present disclosure may be promiscuous or broad-spectrumaffinity reagents that possess a likelihood to interact with (e.g., bindto) more than one polypeptide in a sample. In some cases, the affinityreagents may possess a likelihood to interact with two or more unique,structurally dissimilar proteins in a sample. For example, an affinityreagent may bind with near-equal probability to a particular membraneprotein and a particular cytoplasmic protein based upon a region ofstructural similarity within otherwise dissimilar proteins. In somecases, a binding affinity reagent may possess a likelihood of binding toa particular amino acid epitope or family of epitopes regardless of thesequence context (e.g., amino acid sequence upchain and/or downchainfrom the epitope).

An affinity reagent of the present disclosure may be characterized suchthat it has an identified, determined, or assessed probability-basedbinding profile. An affinity reagent may have the property of binding toa first polypeptide with an identified, determined, or assessed bindingprobability of greater than about 50% (e.g., at least about 50%, 60%,70%, 80%, 90%, 95%, 99%, 99.9%, 99.99%, 99.999% or greater than about99.999%) and binding to a second structurally non-identical polypeptidewith an identified, determined, or assessed binding probability of lessthan about 50% (e.g., no more than about 50%, 40%, 30%, 20%, 10%, 5%,1%, 0.1%, 0.01%, 0.001% or less than about 0.001%). In a particularcase, the difference in observed binding probabilities of the affinityreagent to the first and second polypeptides may be due to the presence,absence, or inaccessibility of a particular epitope or family ofepitopes in either the first or second polypeptide. Probabilisticaffinity reagent binding profiles may be determined or identified by invitro measurements or in silico predictions.

Alternative and Additional Embodiments and Examples

The skilled person will readily recognize the usefulness of thedetectable probes and affinity reagents of the present disclosure,including for example, their tunable avidity and observabilitycharacteristics. The detectable probe or affinity reagent compositionsof the present disclosure may be useful for providing high-resolutionspatial characterizations in micro-scale and single-molecule systems andcontexts. The described detectable probes or affinity reagents may alsoprovide useful characteristics for biological contexts, such asdiagnostic and therapeutic applications.

A detectable probe or affinity reagent of the present disclosure mayfacilitate immunohistochemistry for purposes such as the elucidation ofcellular and tissue structures. Detectable probes or affinity reagentsmay be attached to a plurality of binding components with highspecificity for particular cellular components (e.g., surfacebiomarkers, polysaccharides, structural proteins, organelle proteins,etc.). Detectable probes or affinity reagents may be directly contactedto cellular (e.g., microorganisms) or tissue samples (e.g., tissuesections, frozen tissue samples, formalin-fixed paraffin-embeddedsamples) and incubated for a sufficient time to permit bindinginteractions between detectable probes or affinity reagents and cellularor tissue-related targets to form treated cellular or tissue samples.The treated cellular or tissue samples may be directly observed by anysuitable method (probe dependent—e.g., fluorescence microscopy,scintillation counting, etc.). The detectable probes or affinityreagents may be advantageous for immunohistochemistry applications dueto the high binding avidity and high observability of the detectableprobes or affinity reagents. Analysis of binding data may providehigh-resolution, spatial data on biomarker or biomolecule quantity anddistribution in cellular and/or tissue samples.

A detectable probe or affinity reagent may be utilized as a pull-down orcapture reagent. The high binding avidity may facilitate the capture andretention of targeted species from a heterogeneous mixture of species.An affinity reagent may be free in solution or may be attached to asolid support (e.g., a bead, chip or chromatography resin) during orafter binding to a binding partner. Contacting of an affinity reagentwith a heterogeneous mixture that may include a binding partner maypermit separation of the binding partner from at least one othercomponent of the mixture. For example, an affinity reagent that isattached to a solid support can be contacted with a sample to allow abinding partner to be bound to the solid support via the affinityreagent, the solid support can be separated from the sample, and thesolid support can optionally be washed to remove residual samplecomponents from the solid support. In another example, an affinityreagent can be contacted with a sample to allow a binding partner to bebound to the affinity reagent, the resulting affinity reagent-bindingpartner complex can be attached to a solid support, the solid supportcan be separated from the sample, and the solid support can optionallybe washed to remove residual sample components from the solid support.In either example, subsequent collection and isolation of the affinityreagent or release of the binding partner can effect a separation of thebinding partner from the sample (e.g. heterogeneous mixture).Optionally, one or more binding partners that are captured by an abovemethod can then be detected in a detection assay set forth elsewhereherein. For example, a subset of different polypeptides can be capturedfrom a sample such that other polypeptides from the sample are removedand then the subfraction of polypeptides can be quantified orcharacterized using a polypeptide assay set forth herein.

A detectable probe or affinity reagent may be utilized as a componentwithin a separation system, such as an affinity chromatography column.An affinity chromatography system may be formulated to attach aplurality of detectable probes or affinity reagents with a particularaffinity for one or more binding partners, epitopes, or target moietieswithin a porous matrix or resin. Taking polypeptides as an exemplaryanalyte, a mixture including polypeptides (e.g., a cellular lysate, asample of synthetic proteins or peptides, a non-biological samplecontaining possible biological contamination, etc.) may be applied to acolumn including detectable probes or affinity reagents to affect aseparation between the targeted polypeptides (e.g., those including abinding partner, epitope, or target moiety) and non-targetedpolypeptides. A detectable probe or affinity reagent affinitychromatography system may include a resin or matrix that permanently orirreversibly conjugates the detectable probes or affinity reagents tothe resin or matrix, thereby permitting reuse of the system. Capturedpolypeptides may be eluted from a column after the non-targeted fractionof the mixture has passed out of the column. A detectable probe oraffinity reagent affinity chromatography system may include a resin ormatrix that reversibly attaches the detectable probes or affinityreagents to the resin or matrix, thereby permitting release ofdetectable probes or affinity reagents including captured targets fromthe system. In some cases, an affinity chromatography system may includea detection system (e.g., fluorescence measurement, IR, UV) that permitsdetection of a signal from a detectable probe or affinity reagent as theprobe elutes from the system. The detection system may permitmeasurement of background or unintended release of detectable probes oraffinity reagents from a resin or matrix, as well as monitoring intendedrelease of detectable probes or affinity reagents from a matrix orresin. After capture of target polypeptides from a mixture, polypeptidesmay be eluted from the system by release of the detectable probes oraffinity reagents. In some cases, released detectable probe-polypeptidecomplexes (or affinity reagent-polypeptide complexes) may be collectedthen deposited on a solid support for a polypeptide assay, such as apolypeptide characterization assay or a binding ligand assay.

FIG. 17A-17C depict a scheme for utilizing an affinity reagent as acapture agent. As shown in FIG. 17A, an affinity reagent 1710 isprovided, where the affinity reagent includes a capture handle 1720 anda plurality of binding components with a high binding specificity for atarget binding partner 1730. The capture handle may include any suitablehandle, such as a capture tag (e.g., biotin), a nucleic acid, or afunctional group (e.g., a click functional group). The affinity reagent1710 is contacted with a heterogeneous mixture including the targetbinding partner 1730 and a plurality of non-target species 1735, therebyfacilitating the binding of the affinity reagent 1710 to the targetbinding partner 1730. Optionally, a solid support 1750 including acapture site 1740 that is configured to bind with a capture handle 1720may be present. Capture sites may be generated on the solid support, forexample, as a patterned array or random array. As shown in FIG. 17B, theheterogeneous mixture is separated from affinity reagent 1710—targetbinding partner 1730 complex. If a solid support 1750 is present, theaffinity reagent 1710 and the target binding partner 1730 may becomebound to the solid support at an anchor point 1745 created by thebinding of the capture handle 1720 at the capture site 1740. Depositionof affinity reagents 1710 and target binding partners 1730 on the solidsupport 1750 may generate a random or patterned array of captured targetbinding partners (e.g., target polypeptides). Alternatively, an affinityreagent 1710— target binding partner 1730 complex may be separated froma free solution by a separation method such as affinity chromatography,size exclusion chromatography, gravity sedimentation, filtration,magnetic capture (of a magnetic or paramagnetic solid support) orcentrifugation.

FIG. 17C depicts an optional final step. A captured target bindingpartner 1730 may be subjected to a characterization or quantificationassay utilizing detectable probes 1760 as assay reagents. One or moredetectable probes 1760 may be contacted with the captured target bindingpartners 1760 to characterize presence or absence of a bindinginteraction between the target binding partners 1730 and the detectableprobe 1760. Observation of presence or absence of a detectable signalfrom the detectable probe 1760 may indicate a probability that a bindingpartner, epitope, or target moiety is present at the observed locationon the solid support 1750.

Detectable probes or affinity reagents may be utilized for co-bindingassays. Co-binding assays may refer to any assay where multiple affinityreagents and/or detectable probes are simultaneously contacted with abinding partner, epitope, or target moiety to simultaneously determinethe presence of two or more characteristics (e.g., epitopes) in abinding target. Co-binding assays may be utilized, for example, todetermine the simultaneous presence of two distinct epitopes in apolypeptide, thereby yielding a high-confidence prediction ofpolypeptide identity.

A detectable probe or affinity reagent may be formulated for co-bindingassays by combining two or more unique species of detectable probes oraffinity reagents, each distinguished by a unique detection signature orfingerprint (e.g., first probe uses Alexa-Fluor® 488, second probe usesAlexa-Fluor® 647 fluorescent dyes). Simultaneous detection of uniquesignatures or fingerprints (or lack thereof) at a spatial location canprovide evidence whether one or more detectable probes or affinityreagents has bound a binding partner, epitope, or target moiety at thespatial location.

Alternatively, co-binding assays utilizing detectable probes or affinityreagents may be performed with detectable probes or affinity reagentsincluding nucleic acid barcodes. FIGS. 18A-18C depict a scheme forperforming a co-binding assay utilizing a barcode detection signal. FIG.18A depicts a binding partner 1890 that is contacted with a firstdetectable probe 1810 and a second detectable probe 1840. The firstdetectable probe 1810 is configured to bind with a first epitope ortarget moiety 1870, and includes a first linker 1820 with a barcodesequence 1838 and a terminal priming sequence 1830. The seconddetectable probe 1840 is configured to bind with a second epitope ortarget moiety 1880, and includes a second linker 1850 with a terminalcomplementary priming sequence 1860. If a binding interaction occursbetween the first detectable probe 1810 and the first epitope or targetmoiety 1870, and a binding interaction occurs between the seconddetectable probe 1840 and the second epitope or target moiety 1880, theterminal priming sequence 1830 and the complementary priming sequence1860 may be in sufficient proximity to hybridize by nucleic acid basepairing. As shown in FIG. 18B, a contacted polymerase enzyme 1865 maybind to the hybridized nucleic acids at the priming sequences,permitting an extension reaction to take place. As shown in FIG. 18C,after the extension reaction, the second detectable probe 1840 may bedissociated from the binding partner 1890 after a complementary barcodesequence 1868 has been added to the complementary priming sequence 1860.Subsequent analysis of detectable probe barcodes will detect thetranscribed barcode, thereby indicating that the two detectable probessimultaneously bound a binding partner 1890.

Detectable probes or affinity reagents of the present disclosure may beuseful for medical diagnostic purposes, such as diagnostic assays.Detectable probes or affinity reagents may be applicable in both invitro and in vivo systems. Detectable probes or affinity reagents may beadapted to common in vitro assays such as western blotting and ELISAwith the detectable probes or affinity reagents substituting forantibodies normally used in such assays. Detectable probes or affinityreagents may also be utilized as capture or pull-down reagents forlocating proteins or other biomarkers from fluid or other biologicalsamples. For example, detectable probes or affinity reagents may becontacted with blood or blood plasma to isolate blood-borne biomarkers.Detectable probes or affinity reagents may be utilized for in vivoassays such as PET scanning. In some configurations, in vivo detectableprobes or affinity reagents may be attached to radiolabels (e.g., ¹⁵O,¹⁸F, ⁶⁸Ga, ⁸⁹Zr, ⁸²Rb) to enable the detectable probes or affinityreagents as radiotracers. The detectable probes or affinity reagents mayprovide high-resolution, high signal data for observation of tissuesdisplaying biomarkers for which the detectable probes or affinityreagents are configured to have a high avidity. Likewise, detectableprobes or affinity reagents may be applied as real-time prognostics ordiagnostics for medical procedures. For example, detectable probes oraffinity reagents with a high avidity for a tumor surface biomarker maybe utilized to monitor radiation or surgical treatments in real-time.High-resolution fluorescent data regarding the presence of the surfacebiomarker can provide feedback to surgeons on the clearance of cancermarkers from surgical margins and the progression of the radiation orsurgical treatment (fluorescence extinction would correlate todestruction of residual tumor tissue).

Affinity reagents or detectable probes may be utilized as therapeuticagents for medical applications. High avidity affinity reagents orprobes may constitute logical disruptors or promoters of in vivosignaling and/or binding processes. Affinity reagents or detectableprobes may be attached to a plurality of binding ligands (e.g.,hormones, cytokines, etc.) or binding targets (e.g., surface receptors)to promote an interaction with an in vivo signaling or binding system. Ahigh-avidity affinity reagent or detectable probe may be provided to anin vivo system in sufficient quantity to partially or fully block asignaling or binding receptor system, thereby reducing or increasing acellular response. A high-avidity affinity reagent or detectable probemay be provided to an in vivo system in sufficient quantity to partiallyor fully capture one or more signaling or binding ligands, therebyreducing or increasing a biological response. A high-avidity affinityreagent or detectable probe may be useful as an anti-microbial oranti-viral composition. For example, an affinity reagent or detectableprobe may be configured to bind a receptor system utilized by a microbeor virus to initiate its reproductive cycle. Or, an affinity reagent ordetectable probe may be configured to bind a microbe or virus (e.g., bya viral spike protein), thereby inhibiting the microbe or virus' abilityto bind with a cellular target.

Detectable probes or affinity reagents including alternative bindingcomponents may be useful for therapeutics, diagnostics, or drugdiscovery. Detectable probes or affinity reagents including a pluralityof attached affinity reagent chimeras may be useful for therapeutic ordrug discovery purposes. Affinity reagent chimeras may include anycomplex including an affinity reagent coupled to a secondary molecule,such as a small molecule, nucleic acid, peptide, or protein. Chimerasmay include secondary molecules with specific biological functions, suchas antisense nucleic acids, exons, introns, transcriptional repressors,transcriptional promoters, receptor-binding ligands, enzyme-bindingligands, enzymatic substrates, etc. Affinity reagent chimeras mayinclude aptamer-siRNA chimeras, antibody-siRNA chimeras, aptamer-ligandchimeras, or antibody-ligand chimeras. Detectable probes or affinityreagents including affinity reagent chimeras may be utilized to locate atarget for a secondary molecule, such as binding a target molecule todeliver an antisense RNA to the target molecule. In some configurations,a detectable probe or affinity reagent may include a plurality ofaffinity reagents coupled to the probe, and an additional plurality ofsecondary molecules that are coupled to the detectable probe or affinityreagent.

A detectable probe or affinity reagent may be utilized as a drugdelivery platform. The increased binding avidity of the detectableprobes or affinity reagents provided herein would permit a high degreeof binding to membrane receptors or other targetable systems forcellular uptake. A detectable probes or affinity reagent may beassociated with a drug delivery formulation, such as a colloidalparticle or a coated pharmaceutical formulation. FIGS. 38A-38D depictthe use of detectable probes or affinity reagents for cellular drugdelivery. FIG. 38A shows the combining of detectable probes or affinityreagents including hydrophobic surface modifying groups 3810 with acolloidal drug particle 3820 including a drug formulation 3830. As shownin FIG. 38B, the hydrophobic interactions between the colloidal particle3820 and the hydrophobic modifying groups of the detectable probes oraffinity reagents 3810 form a targeted drug delivery complex. FIG. 38Cdepicts an interaction between a membrane-associated protein 3845 (e.g.,a cellular receptor) embedded within a cell membrane 3842 of a cell 3840and the targeted drug delivery complex due to a binding interactionbetween the membrane-associated protein 3845 and the targeted drugdelivery complex. FIG. 38D depicts the uptake of the drug formulation3830 into the cell 3840 after the targeted drug delivery complex hasinteracted with the targeted membrane-associated protein 3845, therebyreleasing the drug formulation 3830 into the cell 3840. The detectableprobes or affinity reagents 3810 may be released or absorbed into thecell during uptake of the colloidal particle 3820.

Detectable probes or affinity reagents may be useful for characterizingnon-biological materials. High-avidity reagents or probes may beconfigured to bind with non-biological targets (e.g., nanoparticles,polymer structures, etc.). Detectable probes or affinity reagents mayenable high-resolution imaging of structures in non-biologicalmaterials. For example, detectable probes or affinity reagents withavidity for certain nanoparticle structures could be utilized to studythe structure, degradation, and poisoning processes of compositecatalyst materials. Likewise, detectable probes or affinity reagentswith avidity for certain charged surface active sites could be used toprovide high-resolution data on the distribution and availability ofreactive sites on a material surface. Detectable probes or affinityreagents could be utilized to target and locate micro-scale degradationand/or damage in polymer systems, such as synthetic and naturaltextiles. The resolution provided by high-avidity probes may permitidentification of features not apparent to normal optical inspectionprocesses in a wide variety of materials.

Detectable probes or affinity reagents may be applied to additionalpolypeptide characterization methods. Detectable probes or affinityreagents may be configured to enable single-molecule detection Edmandegradation techniques. In general, Edman degradation may producesequence reads for a plurality of peptides by step-wise removal anddetection of single amino-acid residues from each peptide. Asingle-molecule approach to Edman degradation may be facilitated by theuse of detectable probes or affinity reagents to detect a terminal aminoacid or a terminal amino acid sequence. FIGS. 19A-19D depict possibleapproaches to Edman degradation utilizing detectable probes to increasethe signal strength of each sequence read. FIG. 19A depicts a solidsupport 1910 with a plurality of polypeptides bound to the solid support1910. A first polypeptide 1920 has a terminal amino acid residue withsidechain R1 and a second polypeptide 1930 has a terminal amino acidresidue with sidechain R2. FIG. 19B depicts a contacting of theplurality of peptides with a detectable probe composition including aplurality of probe species with high binding specificity for eachnatural terminal amino acid residue (e.g., including at least 20 uniqueprobe species). First polypeptide 1920 is bound by detectable probe 1940which is configured to have a binding specificity for sidechain R1.Second polypeptide 1930 is bound by detectable probe 1950 which isconfigured to have a binding specificity for sidechain R2. Eachdetectable probe of the detectable probe composition has a uniquedetection signature or fingerprint, permitting each binding event to beuniquely identified. FIG. 19C depicts a typical intermediate step in anEdman degradation process after each peptide of the plurality ofpolypeptides has been reacted with an isothiocyanate compound to form acleavable product. A first polypeptide 1925 has a terminal modifiedamino acid residue with sidechain R1 and a second polypeptide 1935 has amodified terminal amino acid residue with sidechain R2. FIG. 19D depictsa contacting of the plurality of modified peptides with a detectableprobe composition including a plurality of probe species with highbinding specificity for each modified natural terminal amino acidresidue (e.g., including at least 20 unique probe species). Firstmodified polypeptide 1925 is bound by detectable probe 1945 which isconfigured to have a binding specificity for sidechain R1. Secondmodified polypeptide 1935 is bound by detectable probe 1955 which isconfigured to have a binding specificity for sidechain R2. Eachdetectable probe of the detectable probe composition has a uniquedetection signature or fingerprint, permitting each binding event to beuniquely identified. In some configurations, detectable probecompositions may be contacted with detectable probe compositions beforeand after modification of the terminal amino acid residues to increasethe confidence of the sequence reads.

The skilled person will readily recognize that many of the embodimentsdiscussed may readily be configured to utilize other techniquesdiscussed above, such as the use of concatenated barcodes as depicted inFIGS. 15A-15D, or the methods for forming weak secondary bindinginteractions like those depicted in FIGS. 8, 9A, and 9B.

EXAMPLES Example 1: Design and Synthesis of Affinity Reagents HavingOrigami Tiles as Retaining Components

Origami tile-based affinity reagents were designed utilizing CADNANOsoftware with accompanying in-house python scripts. Tiles were designedas nucleic acid origami having an approximately square shape with asingle layer. Tiles were designed to have 20 DNA aptamer binding siteson a top face of the tile, and 44 dye molecule attachment sitesdistributed uniformly along the sides of the tile molecule. FIG. 23depicts a schematic representation of the tile-based affinity reagent2310, with dots 2330 representing aptamer attachment locations andcircles 2320 representing dye attachment locations. Tiles were designedto have a side length of approximately 83 nanometers (nm)

Origami tiles were prepared using a scaffold strand of M13single-stranded circular DNA strand containing 7249 nucleotides. 217single-stranded oligonucleotides were designed and synthesized forhybridization with the scaffold strand as ‘staples’ to permit assemblyof the tile-based affinity reagents.

Origami tiles were assembled by combining scaffold strands with the 217oligonucleotides. DNA strands were combined at 90° C. in a buffercontaining 5 mM Tris-HCl, 5 mM NaCl, 1 mM EDTA, 12.5 mM MgCl₂ at pH8.0.The DNA strand mixture was cooled to a temperature of 20° C. and allowedto anneal for 1.5 hrs.

Annealed tile-based affinity reagents were purified via HPLC SizeExclusion Chromatography or 100 kDa size cutoff spin filters. Purifiedtile-based affinity reagents were resuspended in a buffer containing 5mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, 11 mM MgCl₂ at pH 8.0 and stored at4° C.

Example 2: Image Analysis of Origami Tiles

Origami tile-based affinity reagents were prepared as described inExample 1. Tile probes were imaged via transmission electron microscopy(TEM) to confirm the success of the assembly method. Tile probe solutionwas spotted on a glow discharged carbon deposited copper grid andnegatively stained using Uranyl Formate.

Tile probes were imaged on a FEI Tecnai T12 transmission electronmicroscope at magnifications of 30,000× under an acceleration voltage of120 kilovolts (kV). The tile probes appear to have an approximatelysquare shape, with a side length of about 83 nm, as predicted.

Example 3: Design and Synthesis of Tile-Based Affinity Reagents HavingAntibody-Based Binding Components

Antibody tile detectable probes were designed utilizing CADNANO softwarewith accompanying in-house python scripts. The origami tiles weredesigned to have an approximately square shape with a single layer.Tiles were designed to have 6 antibody binding sites (2 on a top faceand 1 along each side of the tile) and 40 dye molecule attachment sitesdistributed uniformly along the sides of the tile molecule. FIG. 25depicts a schematic representation of the tile-based affinity reagent2510, with antibody components 2520 at respective attachment locationsand circles 2530 representing attachment locations for label components.Tiles were designed to have a side length of approximately 83 nanometers(nm)

Origami tiles were prepared using a scaffold strand of M13single-stranded circular DNA strand containing 7249 nucleotides. 217single-stranded oligonucleotides were designed and synthesized forhybridization with the scaffold strand to permit assembly of the tileprobes. 6 of the oligonucleotides were synthesized withtranscyclooctene-modified nucleotides to enable attachment of antibodycomponents via a click reaction.

Tiles were assembled by combining scaffold strands with the 217oligonucleotides. DNA strands were combined at 90° C. in a buffercontaining 5 mM Tris-HCl, 5 mM NaCl, 1 mM EDTA, 12.5 mM MgCl₂ at pH8.0.The DNA strand mixture was cooled to a temperature of 20° C. and allowedto anneal for 1.5 hrs.

Annealed tiles were purified via HPLC Size Exclusion Chromatography or100 kDa size cutoff spin filters. Purified tiles were resuspended in abuffer containing 5 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, 11 mM MgCl₂ atpH 8.0. Antibodies were attached to the tiles via a transcyclooctene(TCO)-methyltetrazine (mTz) click reaction. FIG. 26 depicts an exemplaryreaction scheme for attachment of antibodies (or other bindingcomponents) to the origami tiles. Purified tile lobes with TCO modifiednucleotides were combined with mTz-modified 10× Anti-His tag antibodiesat a temperature of 20° C. and were allowed to react for 6 to 10 hrs.After the click reaction, tile probes were purified via HPLC SizeExclusion Chromatography.

FIG. 27 shows an SDS-page gel for purified antibody-DNA oligo conjugatesgenerated via TCO-mTz click reaction. Lane A contains antibody-DNA oligoconjugate. Lane B contains an mTz-modified antibody negative control.Lane C contains an unmodified antibody control. A dark, uppermost bandis observed, confirming successful attachment of antibodies to DNAoligos.

Example 4: Binding of Tile-Based Affinity Reagents Having Aptamer-BasedBinding Components

Four different species of aptamer tile probe were prepared according tothe method of Example 1. Each species of tile-based affinity reagentcontained a different aptamer with varying binding affinities forterminal histidine tags. The four aptamers used were: B1 (highestaffinity), A1 (moderate affinity), D1 (low affinity), and SC2 (noaffinity). Each tile-based affinity reagent was assembled with 20aptamers and 40 Alexa-Fluor® 647 dye molecules.

Each aptamer was screened for binding against single-molecule arrays ofhis-tagged ubiquitin. Protein arrays were prepared by depositing 10nanogram/microliter solutions of ubiquitin conjugates (ubiquitinattached to a structured nucleic acid particle) on fluidic chips. Eachfluidic chip contained 3 lanes, with each lane having a glass surfacewith a blanket coating of 3-aminopropyl triethoxysilane (APTMS). Threedifferent types of protein arrays were prepared, 1 per lane on eachchip: his-tagged ubiquitin; flag-tagged ubiquitin (negative control);and untagged ubiquitin (negative control). Each deposited proteinconjugate contained an Alexa-Fluor® 488 dye molecule. Protein conjugatelocations in the array were measured via fluorescence microscopy at 488nm excitation. Prior to affinity reagent binding, fluidic chips wereblocked for 60 minutes with a solution containing 1% BSA, 2 nM unlabeledDNA tiles, and 100 mg/ml dextran sulfate in a buffer containing.

To test binding, 15 μl of 20 nM affinity reagent solution was flowedinto a fluidic chip. Probes were allowed to bind for 10 mins. Afteraffinity reagent binding, fluidic chips were rinsed with a rinsesolution. Binding was imaged on the surface using a ThorLab Microscopeat 20× magnification and 647 nm excitation. Microscope images at 647 nmwere compared to images at 488 nm to determine a fraction of proteinsbound by the applied affinity reagents. All four affinity reagentspecies were tested against each of the 3 arrays. Each set of 12experiments was duplicated.

FIGS. 28A-28D depict occupancy rates for binding. Occupancy rate wasdetermined as the ratio of observed locations with binding detected tototal locations with a known protein conjugate. FIG. 28A shows occupancyrates for the B1 aptamer reagents against occupancy rates for the A1aptamer reagents. Despite a lower binding affinity, the A1 aptamerreagents is shown to have nearly as high an occupancy rate as the B1aptamer reagents. FIG. 28B shows occupancy rates for the B1 apatamerreagents against occupancy rates for the D1 aptamer reagents. FIGS. 28Cand 28D compare occupancy for B1 vs. SC2 and D1 vs. SC2, respectively.As shown in FIGS. 28B and 28D, despite its low binding affinity forhis-tagged proteins, the occupancy rate of the D1 aptamer reagents isobserved to be nearly half that of the B1 aptamer reagents and notablyhigher than that of the negative control SC2 aptamer reagents,suggesting that the detectable probes can significantly improve theobserved binding affinity of a weak binding aptamer through an avidityeffect.

Example 5. Binding of Tile-Based Affinity Reagents Having Antibody-BasedBinding Components

Tile-based affinity reagents having antibody-based binding componentswere prepared according to Examples 2 and 3. Each affinity reagent wasprepared with six 10× Anti-His antibodies and 40 Alexa-Fluor® 647 dyemolecules. Binding was tested via the method described in Example 4,utilizing a 0.5 nM probe solution.

FIGS. 29A-29C show fluorescence microscopy images for binding of thereagents. FIGS. 29A and 29B show binding of the reagents to untagged andflag-tagged ubiquitin arrays, respectively. FIG. 29C shows binding ofthe reagents to a his-tagged ubiquitin array. A higher level probebinding to the his-tagged array was observed.

FIG. 30 shows occupancy rates for the affinity reagents against the 3different protein arrays on 6 different chips. High occupancy rates onhis-tagged protein arrays are observed for all 6 chips, while much loweroccupancy rates are observed for the other non-his tagged proteins oneach fluidic chip.

Example 6. Multiplex Binding Assay

Detectable affinity reagents are assembled including either 1 or 2 typesof fluorophore. Available fluorophores have emission in red, yellow,green, or blue wavelengths (R, Y, G, B). Each fluorophore is named toindicate the total number of each fluorophore on the detectable probe.For example, a detectable affinity reagent with 10 red fluorophores and10 yellow fluorophores would be named “R10Y10.” Each affinity reagentwith a unique fluorophore combination is attached to a unique species ofbinding component having a unique binding specificity for a targetmoiety. Two unique pools of fluorophores are created, with compositionsof each pool listed in Table 1 below.

TABLE 1 Pool 1 Pool 2 R10Y10 R20Y10 R20Y20 R10Y20 B10G10 B10G20 G20B20B20G10 B10 B10 G10 G10 R10 R10 Y10 Y10

A pool of affinity reagents is contacted to a first binding partner.Fluorescence intensities are measured at wavelengths corresponding tothe four possible fluorophores. Measured fluorescence intensities arecorrelated to the possible available number of fluorophores. Table 2displays measured fluorophore counts based upon fluorescenceintensities, as well as the combination of bound probes from each poolthat would give rise to the unique combination of observed fluorescenceintensities.

TABLE 2 Observed Binding Intensities (in fluorophores) R Y G B 40 30 010 Possible Observed Probe Combinations Pool 1: R10Y10, R20Y20, R10, B10Pool 2: R10Y20, R20Y10, R10, B10

Based upon the observed fluorescence intensities, available targetmoieties can be predicted for the binding partner.

A pool of affinity reagents is contacted to a second binding partner.Fluorescence intensities are measured at wavelengths corresponding tothe four possible fluorophores. Measured fluorescence intensities arecorrelated to the possible available number of fluorophores. Table 3displays measured fluorophore counts based upon fluorescenceintensities, as well as the combination of bound affinity reagents fromeach pool that would give rise to the unique combination of observedfluorescence intensities.

TABLE 3 Observed Binding Intensities (in fluorophores) R Y G B 30 10 2010 Possible Observed Probe Combinations Pool 1: R10Y10, R10, R10,B10G10, G10 Pool 2: R20Y10, R10, B10G20

Based upon the observed fluorescence intensities, available targetmoieties can be predicted for the binding partner. Rational engineeringof multiplex pools of detectable affinity reagents can give rise tounique binding signatures that predict the existence of multiple targetmoieties within a binding partner simultaneously.

Example 7. Affinity Chromatography

Three affinity chromatography columns are prepared. The firstchromatography column includes a first chromatography resin includingaffinity reagents covalently attached to the resin. The affinityreagents in the first column have a general binding affinity forpolypeptides regardless of amino acid sequence. The secondchromatography column includes a second chromatography resin includingaffinity reagents covalently attached to the resin. The affinityreagents in the second column have a binding affinity for polypeptidesincluding a lysine-arginine-alanine (KRA) amino acid trimer sequencewithin their amino acid sequences. The third chromatography columnincludes a third chromatography resin including affinity reagentscoupled to the resin by oligonucleotide hybridization. The affinityreagents in the third column have a binding affinity for polypeptidesincluding a lysine-glutamic acid-asparagine (KEN) amino acid trimersequence within their amino acid sequences.

The three affinity chromatography column are arranged sequentially. Acrude cellular lysate is applied to the first general separation column.Polypeptides are captured from the crude cellular lysate while thenon-polypeptide fraction of the lysate passes through the column and isdiscarded. After discarding of the non-polypeptide of the lysate, thepolypeptide fraction is eluted by the application of an elution bufferto the first column. The eluted fraction is captured and applied to thesecond KRA-specific chromatography column. A first fraction ofpolypeptides is captured on the KRA-specific resin while a secondfraction passes through the column and is captured. After collection ofthe second fraction from the end of the column, the first fraction ofpolypeptides is eluted from the column by an elution buffer andcollected at the column end.

The first collected polypeptide fraction from the KRA-specificchromatography column is added to the third KEN-specific chromatographycolumn. A first fraction of polypeptides is captured on the KEN-specificresin while a second fraction passes through the column and is captured.After collection of the second fraction from the end of the column, thefirst fraction of polypeptides is eluted from the column by heating thecolumn to melt the oligonucleotide hybridizations, thereby releasing thedetectable probes from the column.

The KEN-specific column is regenerated with a new batch of KEN-specificdetectable affinity reagents to replenish the released affinity reagentsfrom the separation of the first fraction. The separation is thenrepeated for the second collected fraction from the KRA-specific column.A first pass-through fraction is collected, followed by a secondcaptured fraction. After separation of both fractions from theKRA-specific column, a total of four fractions are formed. FIG. 40 showsa schematic of the chromatographic process from the initial cellularlysate to the final four polypeptide fractions, with the preliminarydetermined characteristics of the four fractions listed at the bottom.The preliminary characterizations of the four fractions are evaluatedbased upon the chromatography behaviors of each fraction. Thecharacterizations are considered preliminary due to the possibility offalse negative or false positive capture interactions.

The two fractions of polypeptides captured on the KEN-specific columnare collected as affinity reagent-polypeptide complexes. Each of theaffinity reagent-bound polypeptide fractions is sequentially applied toa patterned silicon solid support including a plurality of attachmentsites, with each site configured to capture and bind an affinityreagent. After each affinity reagent-bound fraction is applied to thechip, fluorescence microscopy is used to measure and record the locationof each occupied attachment site on the solid support.

The two non-captured fractions from the KEN-specific column are attachedto structured nucleic acid particles. Each non-captured fraction isapplied to the silicon solid support, with occupied attachment sitesobserved by fluorescence microscopy as with the captured fractions.After all four fractions are bound to the solid support, a patternedarray of polypeptides has been generated for a polypeptide assay.

Example 8. Synthesis of FluoSphere™-Based Affinity Reagents

Detectable affinity reagents were prepared using highly fluorescentorganic nanoparticles including a modifiable surface chemistry. FIG. 41illustrates a scheme for the preparation of FluoSphere™-based probes. 40nm or 200 nm carboxylate-functionalized FluoSpheres™ (Thermo-Fisher)were modified with polyethylene glycol moieties to form a passivatedlayer surrounding the FluoSphere™ particles. FluoSpheres™ were activatedby NHS-EDC activation prior to functionalization with PEG groups. Thecarboxylate functionalized FluoSpheres™ were combined with a 95:5mixture of NH₂-PEG and NH₂-PEG-azide to form a PEGylated surface coatingaround the FluoSpheres™ containing azide functionalities. PEGylatedFluoSpheres™ were incubated with 3′-dibenzocyclooctene (DBCO)-terminatedoligonucleotides to covalently attach the oligonucleotide to theazide-terminated PEG moieties on the surface of the PEGylated layer. Theincubation occurred in phosphate buffered solution (pH 7.4) overnight atabout 24° C.

Following attachment of the oligonucleotides to the FluoSpheres™,attachment sites were provided for coupling of affinity reagents. Theoligo-functionalized FluoSpheres™ were combined with an oligonucleotideincluding the complementary sequence to the functionalized oligo handle.The mixture was heated to 95° C. for 5 minutes, then annealed at 70° C.for 10 minutes before cooling to room temperature. Alternatively, thecomplementary oligonucleotide could be annealed to theDBCO-functionalized oligonucleotide before attachment to theFluoSpheres™. Sequences for the oligonucleotides are listed in Table 4.

TABLE 4 Oligonucleotide Attachment Handle Sequences  Oligo Name SequenceDBCO-terminated 3′-DBCO-GTTCGTCTTCTGCCGTATGCTCTA-5′ (SEQ ID NO: 8) oligoComplementary 5′-CAA GCA GAA GAC GGC ATA CGA GAT CAT GCT TCC CCA oligoGGG AGA TGG TTT GCC GGT GGG CAG GTT TAG GGT CTGCTC GGG ATT GCG GAG GAA CAT GCG TCG CAA ACG TGTAGA TCT CGG TGG TCG CCG TAT CAT T-3′ (SEQ ID NO: 9)

Example 9. Synthesis of Quantum Dot-Based Affinity Reagents

Detectable affinity reagents were prepared using highly fluorescentinorganic nanoparticles including a modifiable surface chemistry. 15 nmcarboxylated quantum dots (Thermo Fisher) were modified withpolyethylene glycol moieties to form a non-adhering layer surroundingthe quantum dots particles. The quantum dots were combined with a 75:25mixture of NH₂-PEG and NH₂-PEG-azide to form a PEGylated surface coatingaround the quantum dots containing azide functionalities. PEGylatedquantum dots were incubated with 3′-DBCO-terminated oligonucleotides tocovalently attach the oligonucleotide to the azide-terminated PEGmoieties on the surface of the PEGylated layer. The incubation occurredin phosphate buffered solution (pH 7.4) overnight at about 24° C.

Following attachment of the oligonucleotides to the quantum dots,attachment sites were provided for coupling of affinity reagents. Theoligo-functionalized quantum dots were combined with an oligonucleotideincluding the complementary sequence to the functionalized oligo handle.The mixture was heated to 95° C. for 5 minutes, then annealed at 70° C.for 10 minutes before cooling to room temperature. Alternatively, thecomplementary oligonucleotide could be annealed to theDBCO-functionalized oligonucleotide before attachment to the quantumdots. Sequences for the oligonucleotides are listed in Table 4.

Example 10. Optimization of FluoSphere™-Based Affinity Reagents

Various configurations of FluoSphere™-based detectable affinity reagentswere examined to determine an optimal PEGylation strategy. CarboxylatedFluoSpheres™ were PEGylated according to the method described in Example8. Variables tested included PEG size, PEG types, and PEG surfacedensity.

PEGylated FluoSpheres™ were characterized by measurement of Zetapotential to determine the extent of surface passivation. Zeta potentialmeasurements were conducted in 2% phosphate buffer solution (PBS). FIG.42A shows measured Zeta potential for FluoSpheres™ PEGylated withPEG-NH₂, PEG-OH, and NHS-activated FluoSpheres™. A reduction inmagnitude of Zeta potential when reacted with the PEG-NH₂ demonstratessuccessful PEGylation and passivation of the FluoSphere™ surface. FIG.42B shows Zeta potential as a function of the estimated surface densityof PEG groups. FluoSpheres™ were prepared by the method of Example 8with a varied PEG-NH₂ concentration of 0.5 mM, 5.9 mM or 59 mM PEG-NH₂.The Zeta potential is shown to decrease in magnitude as the estimatedPEG surface density increases.

PEGylated FluoSpheres™ were coupled to oligonucleotides to provideattachment sites for coupling binding components. The strategy forproviding attachment sites is described in Example 8. FIG. 42C showsmeasured Zeta potential for PEGylated FluoSpheres™ as oligonucleotidesand attachment oligonucleotides are added to the FluoSphere™. Zetapotential is seen to increase in magnitude as nucleic acids are added,confirming successful coupling of the oligonucleotides to theFluoSpheres™. FIG. 42D shows coupling of aptamer components tooligo-coupled FluoSpheres™ FluoSpheres™ were coupled with poly-Toligonucleotides to form attachment sites that are configured to coupleaptamer components displaying a poly-A annealing sequence. Average totalnumber of annealed oligonucleotides was calculated by qPCR afterannealing of the aptamer components. FIG. 42D shows successful couplingof poly-A-containing aptamer components, and little observed binding ofpoly-T-containing aptamers.

PEGylated FluoSpheres™ were prepared with differing sizes of PEG groups.FIG. 42E shows the measured Zeta potential for FluoSpheres™ PEGylatedwith mPEG-12, mPEG-24, mPEG-36, mPEG-45, or mPEG-112. Zeta potential isseen to decrease in magnitude as the size of the PEG group increases.FIG. 42F shows the polydispersity index (PDI) for PEGylated FluoSpheres™as a function of PEG size. PDI was measured by dynamic light scattering.Beyond a PEG size of mPEG-36, the PDI is observed to increase,suggesting decreased colloidal stability.

Example 11. Binding Characterization of FluoSphere™-Based AffinityReagents

Binding of FluoSphere™-based affinity reagents was tested to assesstheir target-binding characteristics. FluoSphere™-based retainingcomponents were prepared by the method described in Example 8. Aptamerswere pre-annealed with the attachment oligonucleotide at 95° C. for 5minutes, then coupled to the FluoSpheres™. FluoSpheres™ were preparedwith the following aptamers: P7-B1-P5 (target histidine tag), P7-H3T-P5(target histidine tag) and P7-VEGF Aptamer 89 (target VEGF).

Peptide targets for binding studies were prepared by fixing biotinylatedtrimer amino-acid sequences to a streptavidin coated plate. FIG. 43Ashows binding of various aptamer formulations (On-target: P7-B1-P5-FSare FluoSphere™ probes, P7-B1-P5-strep are labeled withstreptavidin-Alexa-Fluor® 647; Off-target: Her2-FS are FluoSphere™probes, Her2-strep are labeled with streptavidin Alexa-Fluor® 647)against various peptide trimer targets. P7-B1-P5-containing FluoSphere™probes are observed to have a similar binding profile to P7-B1-P5-Strepand a dissimilar binding profile to Her2 specific affinity reagents.

Dissociation constants were measured for FluoSphere™-based affinityreagents against various targets. Targets were prepared by couplingbiotinylated targets (protein or peptide) to streptavidin plates. FIG.43B displays binding measurements for P7-B1-P5 aptamer-containingFluoSphere™ affinity reagents against a histidine-tagged Her2 proteintarget. The EC50 was measured to be 6.5 nM, suggesting that theFluoSphere™-based P7-B1-P5 affinity reagents are capable of successfullybinding the histidine tag of the Her2-His conjugate. FIG. 43C displaysbinding measurements for P7-H3T-P5 aptamer against an HHH trimerpeptide. The measured EC50 is 3.4 nM, suggesting that theFluoSphere™-based affinity reagents is capable of binding the trimertarget. FIG. 43D shows measured binding as a function of probeconcentration for a P7-VEGF-Aptamer 89-based FluoSphere™ affinityreagent against a VEGF target (on target) and myoglobin (off target). Amuch higher fluorescence intensity is measured as a function of affinityreagent concentration for binding with VEGF, suggesting that theFluoSphere™-based probe has binding affinity for its intended target.

Example 12. Stability Characterization of FluoSphere™-Based AffinityReagents

The stability of prepared FluoSphere™-based detectable affinity reagentswas tested to determine the binding activity of the affinity reagentsafter extended storage at 4° C. in PBS buffer. Detectable affinityreagents were prepared by the method described in Example 8. Detectableaffinity reagents were prepared with P7-B1-P5 aptamer (target histidinetag). Stability measurements were also performed for single P7-B1-P5aptamers coupled to commercially available fluorescent labelsStreptavidin-Alexa Fluor® 647 Conjugate (Thermo Fisher) andStreptavidin-APC (Thermo Fisher). FIGS. 44A-44C show affinitymeasurements for each detectable affinity reagent at indicatedtimepoints. At each time interval, the affinity of detectable affinityreagents was evaluated as described in Example 11. FluoSphere™-baseddetectable affinity reagents showed a stronger affinity to targets for alonger period of time than the streptavidin conjugates. Surprisingly,the P7-B1-P5 FluoSphere™-based detectable probes have substantiallyimproved stability compared to commercial fluorescent alternative.

Example 13. Binding Characterization of FluoSphere™-Based AffinityReagents

The binding affinity of P7-B1-P5-coupled FluoSphere™-based Affinityreagents was measured in comparison to the binding affinity ofsingle-aptamer P7-B1-P5-fluorophore conjugates. P7-B1-P5-coupledFluoSphere™-based detectable affinity reagents were prepared accordingto the method of Example 8. Single-aptamer P7-B1-P5 conjugates were alsoprepared for the following fluorophores: Streptavidin-Alexa Fluor® 647(Thermo Fisher), Streptavidin-APC (Thermo Fisher), and SureLight™ APC(Columbia Biosciences). Binding curves were measured against a histidine(HHH) peptide using various concentrations of probe or aptamer.Dissociation constants (EC50) were derived from the binding measurementdata.

FIGS. 45A-45D show plots of the dissociation constant for theFluoSphere™-based affinity reagents, Alexa Fluor® aptamers, APCaptamers, and SureLight™ APC aptamers, respectively. TheFluoSphere™-based affinity reagents were measured to have an EC50 ofabout 2.2 nM, The Alexa-Fluor® and APC aptamers had EC50 measurements of28.0 nM and 29.9 nM, respectively. The SureLight™ APC aptamer had ameasured EC50 of 2.3 nM. The FluoSphere™-based affinity reagent appearedto bind the histidine peptide target comparably or better, dependingupon the exact detectable probe system utilized.

Example 14. Binding Characterization of Quantum Dot-Based AffinityReagents

Detectable affinity reagents containing a quantum dot fluorophore as aretaining component were prepared according to the method of Example 9.The quantum dot-based affinity reagents were coupled with P7-B1-P5aptamers (target histidine tag). Binding measurements were made todetermine the binding properties of the quantum dot-based affinityreagents.

FIGS. 24A and 24B display binding measurement data for theP7-B1-P5-quantum dot-based affinity reagents. FIG. 24A displaysfluorescence measurements for quantum dot-based P7-B1-P5 affinityreagents that were contacted with his-tagged Her2 proteins (on-targetbinding) and myoglobin (off-target control). Fluorescent signal isobserved to increase with increasing affinity reagent concentrationagainst the Her2-his target. Little change is observed in thefluorescent signal when affinity reagents are contacted to the myoglobincontrol, suggesting that the quantum-dot based P7-B1-P5 aptamers arecapable of distinguishing and binding their intended target. FIG. 24Bdisplays EC50 measurement data against HHH peptide for the quantumdot-based P7-B1-P5 affinity reagents. The EC50 is measured to be 60 nM,suggesting that the quantum dot-based affinity reagents bind with theirintended target.

Example 15. Synthesis of FluoSphere™-Based Affinity Reagents

FluoSphere™-based affinity reagents with varying ratios offunctionalized to non-functionalized groups were prepared. Binding ofthe FluoSphere™-based affinity reagents was tested to assess an optimalamount of functionalized amount of functionalized groups on the affinityreagent surface. FluoSphere™-based affinity reagents with ratios ofmPEG-amine:amine-PEG-azide of 95:5, 99.5:0.5, 99.95:0.05, and99.995:0.005 were fabricated. After fabrication, the affinity reagentswere attached to B1 aptamers as described in Example 8, and evaluated inplate-based binding studies. The ratio of 95:5 showed about 1.5× highersignal than the ratio of 99.5:0.5 (see FIG. 46 ). An increase in thequantity of available azide groups (the attachment group for theattachment of oligonucleotides to which aptamers were annealed)increases the amount of on-target binding of the affinity reagent whenfully fabricated.

Example 16. Direct Conjugation of Affinity Reagents to FluoSphere™-BasedAffinity Reagents

FluoSphere™-based affinity reagents were prepared via the directconjugation of binding components to the functionalized nanoparticles.B1 aptamers containing a 5′-DBCO functional group were contacted withfunctionalized FluoSpheres™ containing surface-displayed mPEG-azidegroups. Detectable affinity reagents were formed by the reaction ofazide moieties with DBCO moieties. Binding of FluoSphere™-based affinityreagents prepared by the direct conjugation strategy andFluoSphere™-based affinity reagents prepared via oligonucleotideannealing (see Example 8) was compared against a Her2-his tag target.FIG. 47 displays on-target Her2-his binding data for directly-conjugatedaffinity reagents and annealed affinity reagents, as well as binding forboth affinity reagent types against a myoglobin (off-target) control.The observed binding of the directly-conjugated affinity reagents wasobserved to be lower than that of the annealed affinity reagents, butsubstantially exceeded the binding seen against the negative control.

Example 17. Enzymatic Incorporation of Fluorescent dUTP Nucloetides

AlexaFluor-647 dUTP nucleotides were enzymatically incorporated to the3′ end region of an aptamer, as described in FIG. 49B. Fluorophoresincorporated to the aptamers were visualized at 647 nm, as illustratedin FIG. 52 showing successful incorporation of fluorophores.

Example 18. AlexaFluor-647 NHS Ester Conjugation to AminoAllyl dUTP

AAdUTP nucleotides were incorporated into an aptamer, using the methoddescribed in FIG. 49B. The resulting labeled aptamers were chemicallyconjugated to fluorophores, run on a gel, treated with SYBR andvisualized at 488 nm to show doubled stranded DNA (FIG. 53A), and 647 nmto show the presence of fluorophores (FIG. 53B).

Example 19. dsDNA-647 Labeled Aptamer Concentration Measurements

dsDNA-647 labeled aptamer concentration was measured using absorbance at260 nm, the results are shown in FIG. 54 . To examine the conjugationefficiency of AlexaFluor-647 to the aptamers a fluorophore standardcurve was used (FIG. 55A), to determine the fluorophore concentration(FIG. 55B). The Fluorophore concentration measurement was used todetermine the Fluorophore:DNA ratio which estimates the degree oflabeling, as shown in FIG. 56 .

Example 20. dsDNA-647 On-Target Binding and Imaging

To assess on target binding and imaging of a labeled aptamer anexperiment was conducted using a dsDNA-647 labeled aptamer specific tothe epitope HHH. HHH peptides were immobilized on a microplate surfaceand exposed to the labeled aptamer, as shown in FIG. 57A. FIG. 57B showsbinding of the labeled aptamer to the HHH peptide, FIG. 57C shows lackof binding to a negative control peptide, MetGluThr.

To assess imaging the labeled aptamers were visualized on a custom 20 xobjective epifluorescent microscope at various concentrations, as shownin FIG. 57D.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be apparent to those skilled in the art thatsuch embodiments are provided by way of example only. It is not intendedthat the invention be limited by the specific examples provided withinthe specification. While the invention has been described with referenceto the aforementioned specification, the descriptions and illustrationsof the embodiments herein are not meant to be construed in a limitingsense. Numerous variations, changes, and substitutions will now occur tothose skilled in the art without departing from the invention.Furthermore, it shall be understood that all aspects of the inventionare not limited to the specific depictions, configurations or relativeproportions set forth herein which depend upon a variety of conditionsand variables. It should be understood that various alternatives to theembodiments of the invention described herein may be employed inpracticing the invention. It is therefore contemplated that theinvention shall also cover any such alternatives, modifications,variations or equivalents. It is intended that the following claimsdefine the scope of the invention and that methods and structures withinthe scope of these claims and their equivalents be covered thereby.

1.-20. (canceled)
 21. A method of detecting an analyte, comprising: (a)contacting an array of analytes with a detectable probe, wherein thearray of analytes comprises individual analytes each attached to sitesthat are physically isolated from all other analytes in the array ofanalytes, wherein the detectable probe comprises: (i) a retainingcomponent comprising nucleic acid origami, (ii) two or more labelcomponents attached to the retaining component, and (iii) two or morebinding components attached to the retaining component, wherein the twoor more binding components have binding affinity for the same epitope ofthe analyte, wherein the detectable probe binds to the analyte vianon-covalent binding of at least one of the two or more bindingcomponents to the epitope; and (b) acquiring signals from the two ormore label components of the detectable probe bound to the analyte,thereby detecting the analyte.
 22. The method of claim 21, wherein thenucleic acid origami comprises a scaffold nucleic acid and a staplenucleic acid hybridized to two regions of the scaffold nucleic acid,wherein the two regions of the scaffold nucleic acid are separated by anintervening region that does not hybridize to the staple nucleic acid.23. The method of claim 22, wherein the scaffold nucleic acid comprisesa circular nucleic acid.
 24. The method of claim 22, wherein the nucleicacid origami comprises a plurality of staple nucleic acids hybridized tothe scaffold nucleic acid.
 25. The method of claim 21, wherein the twoor more label components comprise fluorophores that emit fluorescence atthe same wavelength, and wherein the signals comprise the fluorescence.26. The method of claim 25, wherein the retaining component maintainsthe at least two label components at a spacing of at least 15 nm. 27.The method of claim 21, wherein the analyte is attached to the site viaa structured nucleic acid particle.
 28. The method of claim 21, whereinthe analyte comprises a polypeptide.
 29. The method of claim 28, whereinthe epitope is a trimer amino acid sequence.
 30. The method of claim 28,wherein the epitope is a tetramer amino acid sequence.
 31. The method ofclaim 28, wherein the polypeptide is denatured.
 32. The method of claim21, wherein the two or more binding components comprise antibodies orfunctional fragments thereof.
 33. The method of claim 21, wherein thetwo or more binding components comprise nucleic acid aptamers.
 34. Themethod of claim 21, wherein the structures of the two or more bindingcomponents are the same.
 35. The method of claim 21, wherein the sitesare separated by at least 100 nm.
 36. The method of claim 21, whereinthe detectable probe is bound to the analyte via non-covalent binding ofonly one binding component of the two or more binding components to theepitope.
 37. The method of claim 21, wherein the retaining componentcomprises a first nucleic acid sequence that is complementary to anucleic acid that is coupled to one of the binding components.
 38. Themethod of claim 37, wherein the retaining component further comprises asecond nucleic acid sequence that is complementary to a nucleic acidthat is coupled to one of the label components, wherein the firstnucleic acid sequence differs from the second nucleic acid sequence. 39.The method of claim 21, wherein the array of analytes comprises at least1×10⁴ different analytes each attached to sites that are physicallyisolated from all other analytes in the array of analytes.
 40. Themethod of claim 21, wherein the structures of the two or more labelcomponents are the same.
 41. The method of claim 21, further comprising:(c) contacting the array of analytes with a second detectable probe,wherein the second detectable probe comprises: (i) a second retainingcomponent comprising nucleic acid origami, (ii) a second label componentattached to the second retaining component, and (iii) a second bindingcomponent attached to the second retaining component, wherein the secondbinding component of the second detectable probe is different from thetwo or more binding components of the detectable probe, wherein thesecond detectable probe binds to the analyte via non-covalent binding ofthe second binding component to an epitope of the analyte; and (d)acquiring signals from the second label component of the seconddetectable probe bound to the analyte, thereby detecting the analyte.42. The method of claim 41, further comprising, prior to step (c),removing the detectable probe of step (a) from the analyte.
 43. Themethod of claim 42, wherein the analyte comprises a polypeptide andwherein the method further comprises (e) identifying the polypeptidebased on the detecting of steps (b) and (d).